CA2081203A1 - Method for enriching fetal cells from maternal blood - Google Patents

Abstract

A method is provided for enriching fetal nucleated, erythroid cells from maternal blood comprising the steps of: (a) incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of the ligand to the cells; (b) removing unbound blood products; and (c) incubating the bound cells in the presence of erythropoietin such that fetal cells are preferentially enriched.

Description

2~2~3 wo 91/16452 PCr/US9ltO2789 De~nD~on A MEIHOD FOI~ E~RI`TG ~ETAL CELI S
~:ROM MATERNAL BLOOD
s Tec~-cal Field Thc ~ren~on re~ates to a me~od of ennc~ing fetal cells ~om matcsDal blood.

10 Back~round o~ the InventiQn Appro~na~e~y S3 mi~lion wome~l become pre~ant ~ the United States yearly, resuldng in 3.8 millio~ deliveries. lhere are an addidonal 10 million dcl~renes in the other afEluent countncs in the world. Prenatal tes~g is used i~ a sub~oup of these women who ha~e a significant risk of having a fetus wi~ a 15 genetic disorder such as Down's Syndrome.
A~ prese~, the only way of diagnosing fetal d~sorders is to obtain cells from the amnioic fluid (amnioce~tesis~ or ~e suIface of the fetal sac (chorioDicv~lus) of ~e mo~cr. T~ese procedures are ~peDs~e a~d carry a risk of spontaneous a~ordon of bctween Y2q~0-l ~o. BeG~se of uhe risk of s~ontanecus 20 abomon, thesc collecion procedures are recommended only for women who are at a big}l-~isk of ~ a c~ild wi~ a geslesic defec~ For example, only wome~
ovcr age 35 are a~ ed to have the test beca thc risl~ of Dow~'s Sgndrome ss ~ighcr in tha~ group. S~me of thesc women refuse the test beca~lse of the Ask ofspontaneous abomon. Even ~ough many of these high-risk women are tested, 25 only a frac~ion of fetuses with Down's Sy~drome are dese~ed. The high-ns~c women rcpresent such a srnall por~ion of the women ha~iIlg childre~ tha~ the low-ns3c population still delivers most of the afELic~ed childre~ Eighty percent of those c~ildren born wish Oown's Syndrome are ~rom the ~low-ns~," under 35-year-old gr~up. This situa~on is also true of many othcr genctic defcas or disordcrs.
3û It is thercfore dcsir~blc to pr~de a test that would resolve this ~g dilemma by pro~iding a safe mcthod which could be ava~able to all - pregna~l womeD, irrespcctil~e of nsk hclor al~d withou~ nsk of spontaneous a~ortio~ Although it is know~l that fetal cells circ~late in the blood stream ofpregnant women (sce ~lozik asd Pawlountz~ Fetal Cel~s isl thc Mater~l 35 Circula~on: Daection by Dircct AFP-Imm2~0fluorescence," Human Genencs ~1-224, 1982), fctal cclls arc prescnt in such low conccntra~ions that the proccdures neccssa~y to iso1atc thcm asc a~tsemc~y dif~cult and timc~o~usuming.

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2û8i2d~
wo 91/16452 PCI/US9l/0278~

For e~ample, Hcrszenberg et aL ("Fctal Cells in the Blood of PregDaut Womcn:
De~ecnon and E~nchment by Fluorcscence~ va~ed Cell Sor~ng," ~Q~,~L
1453-14S5, March 1979) used a Fluorescellce-Ac~vaud Cell Sorter (FACS) iD order to detect fetal cclls in ma~esDal blood. T~e procedure 5 used, howe~ver, is not adaptablc to routinc clinical tes~g proc~dures, in part, due to the grea~ acpcnse and e~e req!~ired to rus a FACS machi:~e. The me~hod of Henzenberg et aL is also dcficicnt for rout~e c~inical testing because it rc~ures dctermination of ~A t~ Bianchi ct aL (~irc~ Hybridiza~on to DNA From Small ~lu n~ers of Flow-Sortcd Nucleated Ncwborn Cells,~ C~omem 10 ~:197-202, 1987) also used a FACS machinc to detect lluclcated cells, although the blood sample was not obtained ~om the mother but from the newbor~'s umbilical cord.
The prese~t inverltion provides a method for emiching fetal cells that overcomes these disadsantages, and ~her provides other rclated 15 ~ges SummaI~r of the Invcntion The present i~Ivcntion is directet toward methods for e~nching fetal mlclca~ed, e~roid cells from matcrnal blood. Within onc aspect of the present 20 invention, such a method comprises the steps of ~ incllbaD~g a sa~le of mate~al blood with an immobilized ligand capable of specifically binding to fetal m~cleated, e~hroid cells under condidoDs and for a ~me suf~cient to allow sp~cific binding of the ligand to the cclls; rcmo~ring unbound blood products; and inalbating the bound cclls in the presence of crythropoietin such that ~e fetal 25 cells are preferentially eDriched. Within one embodiment of this aSpect of the im~en~on, the immobilized liga~d is an immobjlized antibody.
Within aIlother aspect of the ~ention, a method is pro~ided for enriching fetal n:ucleated, c~roid cells ~om mater~al blood, comprising the s~cps of.- incuba~ing a sample of mater~al blood with a fi~t member chcmically 30 linked to a ligand ca~ablc of spccifically bindi~g to fetal mlcleated, csythroid cells u~der conditsoss a~d for a ~me su~cient to allow specific bi~ding of thc Iigand to thc ce~ls; adsorbing the cclls to aD immobilized second member, the secand membcr being capablc of binding to the first member ~vith an affinity cons~t of grcatcr than a~out 108 M-1; rc~nng unbau~d bload produc~s; nd ina~bating 35 the bound cells in t~e presesce of esythsopoic~ such that the fetal cells arepre~ferentially cnrichcd Suitable first member~eco~d mcmbcr bindmg pairs iI~clude bio~in-avidin, bio~reptavidin, biocytin-aYidin, biocyon-streptavidin.

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20,~12~3 wo 9~ 2 Pcr/US9l/02789 methotrexate di~ydrofolale reduc~asc, 5-~uorouracil-thimydylate synthetase, and nboflavin-ribo~avin binding protei~ Within one embodiment of this aspea of the i~vention, the first member which is chemically lillked to a ligand is a biotinylated ar~t~body aDd thc ~obilized sccond D~cmber is ~obilized a~ndiIL
Within a~othcr aspea of the present imrcntion, the method comprises thc steps of: ina~ a c~mplc of maternal blood with a fi~st ligand ca~able of spe~ifically binding to fetal n~lcleated, e2ythroid cells under conditions ant for a timc su~fiacnt to allow spe~:ific binding of the firs~ ligaDd to the ceLs, incubating the sample with a first memoer chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand; ads~rbi~g the cells to an immobilized second member, the second member capable of binding to the first member with an ~f~nity coustant of greater than about 108 ~1; removing unbound blood products; and incuba~ng the bound ce~ls i~ the presence of esyt~opoiesII suc~ tha~ thc fetal ce~ls are preferentially erlric~c~ Within one embodiment, thc first ligand is an an~ody which specifical~y binds tO fesal mlcleated, erythroid cells. Within preferred embodiments, the firsl memberwhich is chcmically lini;ed to the se ond ligand is a bio~rlated ant~o~y. In such an cm~diment, thc immobilizcd secbnd member is immobi~zed a~ndilL
As an alternative to the step of incubating bound cells in the presence of ~ropoiet~, another aspect of the present invention comprises the steps of. (a) i~cuba~g ~e bou~d ce~3s ~nth ammonia and chloride ions and a car~o~iG a~drase inhibitor under coudiions a~d for a ~ime su Eicie~t to allow accumulauon of ammonium ions within the bound cells; and (b) incubating the ~ou$ld cells contai~ing aca~mulated ammonium ioDs in the presence of ammonia and carbon dio~de such that selective hemolysis of maternal blood cells occurs I~ addition, the present ilIvention may also be pesformed using a combination ofbath erythropoietin e~richment and the me~hod described abo~re wherein the cellsare incubated with ammonia and chloridc io~s and a carbo~ic an~drase i~hibitor 30 undcr condi~ions and for a ~me su~aent to allow accumlllaion of ammonium ioDs within the bound cells, followed by the i~cubation of bou~d cells containing accumulated arnmoDium ions in the presence of ammo~ia and carbon dioxide such that sclecnve hemoly~is of ma~c~nal blood cc)ls occurs. These two enric~me~t mcthods may be performed se~uc~tially, and in either order.
Within anothcr aspect of shc present inveruioD, a mc~hod is pr~idcd for cnriching fctal ~ucleatcd~ c~hroid cells from matc~al blood, comprising the steps of: incubating a sample of matcrnal bload in the presenci- of 2~81203 -wo 91/~6452 PCr~US91/02789 esythropoietin such that the fct I celLs are enriched; incuba~g the ennched cells with an immobilizcd liga~d capablc of speQfically bmding to fetal ~ucleated, crgthroid cells under conditions and for a time sufflcient to allow specific bindiIIg of the li~d to the cells; and remo~ring unbound blood producrs. Altcsnatively, wi~hin another embodimes~t of t~e present invention, the enric~ed cells may be =ob~izcd by incuba~g tbem ~th a f~st member chemically lin~ed to a ligand capablc of specifically binding to thc cells under conditions and f~r a time su~cient to allow specific binding of the ligand to the ccl~ and adsorbing the cclls to an immob~ized second membcr, the sccond membcr being capable ~f 10 binding to the first mcmber with an affinity constant of ~eater than about 108 ~1 Within yet a further embodiment of the present i~vendon, the enriched ceL~s may be =obilized by incuba~ng thc e~riched cell~ with a first ligand capable of speci~ically binding to the cells uIIder conditions and for a time su~icient to allow s~ecific binding of the first ligand to the c lls; incllbating the 15 sa~le with a fi~t member chemiG~lly linlccd to a second ligand G~able of ~ically binding to the first ligand u~ldcr condi~o~s and for a time sufficient to allow the sccont ligand to bind to ~e 5rst liga~d; a~d adsorbing the cells to a~immobi;lizcd second member, the second member being capable of b~ding tO the fi~st membcr with an ~nity coDstant of greater than about 108 ~1. Suitable 20 combinations of first and se~ond ligand are discussed in detail below.
In addision, within thc aspccts discussed above, the methods may fanher comprise (subsequent to removing the unbound blood producls) the sseps of incuba~ing the bound cells with ammonia and chloride ions and a carbonic an~rdrase inhibitor under conditioDs and for a time sufEicient to allow 25 ac~2mulation of ammonium ions within the bound cells; and incubating she cells c~nt~ining accumulated ammonium ions in the presence of ammonia a~d carbon *ioxide, such that selecti~re hemolysis of maters~al blood cells occurs.
Within other aspe~s of the present inventio4 as an alternative to first i~lcubating a sample of maternal blood in the presence of esythropoicti~, the cell~s are incubatcd with ammonia and chloride io~ arld a carbonic anhydrase inhibitor uIlder conditions aIId for a time su~cient tO allow aa:umulauon of ammoninm ions within thc bound cclls, followed ~y incubanon of the cells cotlta~ing acNmulated ammoDium ions in the preseslce of =onia and carbon dioxide, such that selccn~ e hemolysis of ma~ al blood cells occurs.
Withi~ the present invention, a variety of ligands may be utilized.
includiI~g antlbodies, esythropoietin, and transferrin. The ligand may be immobilizcd on any of a ~rariety of solid supports, such as hollow fibers, beads, 2~3~203 wo 9It16452 PC~tUS91tO2789 magne~c beads~ pla~es, dishes, ~asks, meshes, scre~s, solid ~bers, membranes, and dips~clcs.
Wi~hin another aspect of the present im~c~io~ a method is provided for typ~ chromosomcs of fctal mldeated ery~roid cells, comp~ the steps of:
S ino~basing t~e fesal nucleated crgt~roid cclls in a media c~Dsai~g esy~opoie~
under conditions and for a ~mc su~cient to i~ucc mesaphasc ill the cells; fi~ng the D~A of the cells; staining the fixed DNA suc~ that chromosomes may be obses~red; and ~nining ~c sta~et DNA thereby allawing ~c ~rping of the c~romosomes.
These and other aspccts of the present invention will become e~rident upon reference to the following detailed description.

Detailed Descn~ion of ~e Tnvention Thc present inv~nion provides methods for the cnnchment of fetal 15 mlcleated, erythroid cclls from mater~al blood. ~ta~l blo<~ tains, among man~ other ~pes of cells, both adult and fe~al nudca~ed, ery~roid cells. Throug~thc efforts of the present in~emion, fctal nuc~eated, e~hroid cel~s may be cI~nched from as few as 1 in 106 in maternal blood, to an emiched concentration of about 1 i~ 103, and prefesa~ly, to a out 1 in 102. With n the comext of she 20 present invention, nucleated csy~roid cells contain a nuclels and generally includc ery~hroblasts as wcll as other cr~rthroid prccursor cells. : ~ -Materual blood may be obtained from a prc~ant female using conventional techniques we~l known m the art. Preferably, penphera~ blood is ~:
drawn from an casily obrainable source such as the alltecnbital vein (she arm ~ein) 25 with convcntional venipullcmre techni~ues. Once the masernal blood has been drawn, it may be frozesl USiDg co~venno~l techIlique$ or stored as 4C for a ma~mum of 4 to 7 day~. VaIious a~ticoagulants may be added to the blood as ~eccssary, including, among othcrs, ACD, C~DA~ EDTA, and Heparin.
The matcsnal blood is then subjected to a selecnon method in 30 accordancc with thc present in~e~tioD, wherei~ prefcrentially ennched fe~al cells may be obtaincd without the need for further purification. for example, by a Fluorescence-Acsvated CelI Sorter (FACS). In general, the methods of the prescnt in~ention comp~isc the stcps of: (1) incubati~lg thc matcrnal blood witheithcr an immobilizcd ligand, or a ligand which w~l su~sequcntly be immobilized.35 such that ~e ligand binds to and hcnc~ immobilizes the fetal ~lucleated, erythroid c~lls; (2) rcmoving ~ound blood produas; and (3) prefereunally cmichiDg the bound cc~ls for fetal nucleated, e~hroid cells. As noted above, these basic steps .... . .. .
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20~ i20 ~ -wo 91/l6452 Pcr/US91/0278 may be perfo~ned in an altc~aiYc order, for example, comprismg the steps of:
(1) preferen~ally enriching mate~al blood for fctal nuclealed, erythroid cells;
(2) incubating the enriched cclls with either a~ imrnobilized ligand, or a ligand w~ will subsc~uen~y be immobilized, such t~at thc ligand binds to a~d he~ce S immobilizes thc fetal nuclca~ed, crythroid cclls; and (3) rem~ unbound blood products. Ihc presemt im~en~ion may be pcrformed uilizing a de~ice as descr~bed in a related appUcation entitlcd ~noselection Device and Method", attorney's toc3ces ~umbes 2~ao72.401, w~ich is mc~sposa~cd hese~ ~y refesence.
~Ithin onc aspcct of thc present im~ention, ~e maternal blood is 10 incubated with aII immobilized ligand capable of specifically binding to fetal nucleated~ erythroid cells under conditioDs, a~d for a time sufEiaent to allow binding of the cells to the ligand. Gencrally, incuba~on of about 15 to 30 miuutes at 4~C to 37C is preferreL If the incubation step occ~rs as the ce~ls are passed over a column, thc flow rate shoùld be sufEciently slow to allow the cells ~o binL
15 Preferably, t~c cells should bc allowed as least 15 m~utes n w~ich to traYerse the bed of thc colum~L
As noted above, the ligand should be chosen such that it is capable of specifically bi~ g fctal nucleased, esythroid cells. Within the context of the present in~,reution, the ligand is defiIled to be "spe~ically binding" if it is capable 20 of binding to fet 1 nucleated, ery~hroid ce31s, but ~ot to more than about 10% of the ma~ernal blo~d cells. Ihe rcla~ve percentage of bound fetal nucleated, csy~roid c~lls to other cetls may be rcadily teterminet by ~ ysis w~th a speci~cmarlcer such as Alpha Fetal Protein (AFP). For example, at a~y poin$ within the methods discussed below, the relative percentage of fetal cells to maternal cells 25 m~y be determined with either glucose o~dase or ~uoresceirl eonjugated an~-AFP ~tibody (see A. Kulozi3c and L ~L Pawlowitzl~i, ''Fetal Cells ln the Materna~
Circula~iorL Detection by Direa AFP-Immunofluoresc~nce," .Fl~nan Genen ~ ~21, 1982). ~his determ~ation is preferably perf~rmed after two or more plln~Sca~ion steps.
ligands which s~ecifically bind to fetal nucleated, e~hroid cells are Icr~own in the art9 induding cr~hropoiesin (Amgen, Thousand Oaks, Calif), transfcrIin (Sigma Chcmical Co., Sn Louis, Mo.) and selected an~odies.
Monoclonal an~bodies which specifiGIlly rec~gI~izc nucleated ersrthroid cc~ls are partiallarly prcfer~e~ Monoclonal a~n~odies to nucleaud cry~hroid cclls such as 35 arld-transfernn reccptor an~bodies may be obtained fsom con~cntion I suppliers (Becton Dic~son Immunoqtomcrry Systcms, Mountain ViCW, ~lif ).
Altcrl~atively, monoclonal anti~rythroid antibc~dies, such as EP-1, may be 2~12~3 wo gl/16452 pcr/us91/o2789 produced using tec~niques well known is ~e art ~see Yoko~i et al, "Monoclonal Ann~odics Detc~ng A~igenic Dcte~ts With Res~ictcd Expressio~ On Ery~hroid Cells: From thc Erythroid Commirted Progenitor Level to the h~amre E~soblast,~ I376, 1984; sce also Heddy Zola (ed.), ~1 S C~C Prcss, Boca R~ton, Fla, 1987).
Brief~r, cells may be genaated for immulliz~ion ~om fctal li~.~er clonal esy~hroid allturcs, and e~nchcd for progenitor ce~ls. Thu~, thc populatio~ of cells used for a~gens and for pr~y sc~e~g may co~tain immacure esy~hroblasts, erythroblasts of an inte~med~atc dc~ee of man~ r, aud presumably, proge~itor 10 cclls of BFU-E and CFU-E typcs. ~hesc ccDs may be used for intravenous immani~tion~ followcd by removal of the spleen and fusion of the spleen cells with a myeloma line such as NSL using standard techniques. The resulting fused cells, or hybridomas, may thcn bc screcncd against thc above~escn~ed cells ~g convcntional te~iqucs (sec Yokochi e2 al., ~pr~).
Thc entire specificalh~r binding antibody need not be used as the ligand. More specifically, only the binding region of the antl~ody is necessary to spccifically bind fetal nucleated, erythroid cclL~ Ihus, an~body ~agme~ts such as Fab or F(ab')2 ~a~en~ may bc used within the present imrendoD. Additionally, the binding regions of the specifically binding a~tl~ody may be incorporated into a - 20 ~ew protein, which may be ~sed as thc ligalld (see Reic~ n et aL, 'lResha~in.g Human Andbodics For ~herapy," arure ~ 323-327, 1988; Verhoeyen et al., ~R~shap~ Human Antl~odics: Graf~ng an Antilysoyme Acnvity," Science ;2~2:15341536, 1989; and Rober~s es al., "Gesesation of an A~tibody with Enha~lced Af~i2y and Spccificity for its Antigen by Protein E~lgineering," ~Jan~re 31-734, 19~7).
Within the present invention, the ligand is immobilized i~ order to allow separa~on of bound cells from other blood produc~s. Many suitable suppons re wcll known in thc art and includc, among others, hollow fi~ers (Amicon Corpora~on, Dam~ers, Mass.), beads (Polysciences, War~i~gto~, Penn ), 30 magnctic bcads (Robbin Sciendfis Mountain View, Calif), plates, dishes and flas~ (Co~mng Glass Wor~cs, ColDing, N~Y~)s mcshcs (Bec~on Dicl~nso~, Mounta~ Vicw, Cali), screens and solid fibcrs (see Edelmarl et al U.S. Patent No. 3,843,324; see also Kuscda et aL, U.S. Patcnt No. 4,416,m), membra~es (~mli~orc Corp Bcdford, Ma~s.), and dips~cl~ A ~ariey of di~Eercnt sources 35 e~ for supparts other than thosc designated. Par~a~larly preferred is a support suc~ as Biogel P 60~ (BIORAD, Ric~mond, Cali). Biogel P 60~ is a porous polyacrylamidc hydrogel bead Thc beads are genes~lly sphenG~l, on average ,-.- . . .. .: ....... . .. .
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20gl2~'., wo 91/16452 PCl/US91/0278 a~out 250 micro~s in size, and havc an average pore sue which excludes moles:ules larger than appro~mately 60,000 daltoDs.
A variey of methods may be used to immobilize thc ligand onto a support. For ~ple, a ligalld, such as an antlbody, may bc directly ooupled to S t~c support by various methods ~rcll lcclown in the art (sce J. K Inman, Metl~ods ~_ VoL 34, ~_~ W. B.
Jakoby and M. Wilc~el~ (eds.), A~:ademic Press, New Yor~, p. 30, 1974; sce also M. Wilche~, and W. Bayer, ~Ihc A~idin Bio~in C omplex in BioaI~alytical Applications,~ Analvt. Biochem. t7t:1-32, l988). ~esc mcthods include the usc 10 of glutaraldeir~rde, carbodiimide, carbonyl diimida~ole, cyanoge~ brom~de, tosyl chloride, biotin/a~ridin, and bio~in/streptavidin. Once thc ligand h~c been immobil~ed onto the support, the maternal blood may be incubated with the immobilized ligand under conditio~s, and for a time sufEicient to allow binding of t~e ligand to thc cclls. Within ~hc context of the present i~e~on, suitable 15 conditions for binding to occmr include inalbation in a physiological bu~Ecr as about 4C to about 3~C Partic~Ilarly prefe~red temperan~res range from about 4C to ~m temperanIre. T~c time of iIlalbanon depends on the afflnity and a~dity of the ligand for thc ce~, alld may be readily dete2mined. Generally, incubation for abolIt 15 minutes to one hour is prefcrred. Following incuba~on, 20 urlbou~d blood prod~ s may be removed, a~d fetal cells enriched usmg the mcthods d~scribcd herein.
Within anothcr aspca of thc present inven~on, a sample of maurnal blood is incubated under suitable condiions with a ligand which is chcmically li$11:ed to a fi~st member, aDd then adsorbed to a second member 25 which LS immobilized on a solid su~por~ The fi~st member should be capable ofbinding to the second member with an affinity of grcater than about 108 M-1.
Many sl~itable first member-second member binding pairs are well know~ in the art. Ihese include, amo~g o~ers, bio~-a~ndin, bio~n-strepta~ridi~, bioc~-aYidin, biocyan-strcpuYidin, mcthotrexate dihydrofolatc reduc~asc, 5-Buorouracil-30 t~dylate synthetasc, n~oflaYill-n~ofla~in binding protein, a~tl~ody-protem A,and an~;bod!y-protein G. T-l a preferred embodimerlt, the first member is biotinand thc scco~d member is a~idin Eithcr member of thc above des~s~bed bind~g pairs may fuIl~on as the second mcmbcr, with the complemema~y member fimctio~g as the first 35 mcmber. Furthcrmore, combiDa~ons of the first member - sccond member binding pair may be employed. For ex~le, bio~ m2y be linked to thc ligand, as well as adsorbcd ~o ~c suppor~ The cell, ligand, biotin~omplex and bio~

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2~8~203 wo gl/16452 PCr/US9l/02789 support~omplex may thcn be bound together through an i~ bation step with avidin. A~.~idin is mul~/alcnt, permitting the formation of a cell, ligand, biotin, avidiL biotin, support~omplex which immobilizes the celL
Within one exa~nple of ~is embodi~cnt, a samplc of mater~
S blood is incubated wi~h a bio~mylated an~l~ody undcr condisions and for a t~e su~iQcnt to allow binding to ocQlr. T~e sam~le is then incubaud with, or passed o~rer a support which co~tains immobilized a~idin Cclls which are coupled to thebioti yla;ted a~l~body are atsorbcd to the immobilizcd avidin, thus allowing scpaIation of ce~ls ~om uIlbound blood produc~. Subsc~ucntly, u~bound blood 10 products may be removed, and fetal cells enrichcd using methods descn~ed below.
Within yet another qspect of the present irlveIltian, a two-step method is used to immobilize the fetal nucleated, ery~roid cells. Brie~y, a first ~igand ~s inalba~cd with a samp~e of thc matcrnal blood undcr sLutable conditions as descnbed above. Subseque~tly, a second ligand which has becn chemically 15 lis~ked to a first mcmber is atded. T~e second ligand is capa~le of binding to the first ligand. T~e celL fiIst ligand, sccond 1igand, fi~st mcmber-complex may ~enbc adsorbcd onto an immobilized sec~nd membcr. tlrus allowing thc separation of cclls from unbound blood products. Reprcsenutivc examples of thc first member-sccond member binding pair ha~rc been disa~ssed abovc. Representative 20 ac~mples of the first liga~d includc ery~ropoietin, transferrin, and sele~ed antibodies. Once thc first ligand has been selected, the sc~ond ligand is chosensuch that it spccifically recogr~izes a~d binds to the first ligan~ Within a prefcrred embodiment, ~e sccond ligand is an antibody, ~or ~amplc an anti~ry~ropo-eti~
(Terry Fox I~boratory, Vancouver, B.C., Canada), a~lti-~ansfer~in (Chemicon 25 Intl., Inc~, Temecula, Calif ), or and-immunoglobulin antibody. Anti-immunoglobulin antibodies may be prepared using techni~ues well Imown i~ the art, or may be obtained from con~lentional sources, includiIIg, among others, Sigma Chemical Co., St. L:ouis, MOq and Becton Dic3cinson ImmlL~ocytomesry Sy~tems, Mountai~ View, Calif.
Wit~in a preferred embodiment, the ~st ligand is an an~body wbich specifically recog~izes fetal nucleated, erythroid cells such as an anti-t~ansfe~in receptor an~body (Becton Dic3ciDson Immunocytometry Systems, Mouma~ Vicw, Calif.). The a~tl~ody is malbated with a sample of maser~
blood A bio~yla~ed ani-Gunoglobulin anibody, such as bioinylated goa 35 a~d-mouse IgG (the second ligand which is chemically linlccd to a first mcmber) is thc~ added and incubaud with the sample. The sample is then incubatcd wi~, or passed over a bed of material which con~ s the immobilized second member. in '`
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208120`3 wo 91/16452 Pcr/US91/02789 this G~c"mmobilizcd avidin. Thc cClL antibody, an~i~ moglobulin anibody, and bio~in-complex will be adsorbed to the immobilized avidi~L thus allow~ng thesubsequent removal of unbouIld blood products.
As notcd abovc, oncc the ce~ls have been immobilizcd, unbou~d 5 blood products may be rcmoved. In onc cmbodimenr, the immobilizçd cells are nnsed with a physiological bufEer, thcreby removing thc unbound blood produ~s.
Various methods may be uscd to ri~se the immobilized ce11s, deperlding upon the gpe of suppor~ choscn. T~cse mcthods ~clude, among o~crs: washing or flushing the sup~r~ magnctiG~y a~ ng thc support out of so1u~on, followed 10 by rcsuspension m a physiological buffer, and centrifuga~ion followed by res~ension. Various physiological buffers are also well l~own in the art, induding PBS, PBS plus albumin, such as Bovine Serum Albumin (BSA), norm~
salinc, and ccll culture media.
Once u~ound blood products have been removed, baund ce~ y 15 bc prcfcresl~a11y ennchcd for fetal nuc3eatcd, erythroid cells. As noted above, at least two alterrlative methods may be used either separa~ely, or together. If the two methods arc performed togcther, cither method may ~e performed first.
W~l~in one embo~mcnt shc ~ound cclls are cul~red under selected a~lture conditions in ~c prcsencc of es~hropoietul (Amgen, Thous~d Oalcs, Cali~) ~see 20 Emewn ct aL, ~Developmental Regulatio~ of ~ropoiesis by Hematopoie~c Growth Factors: Analysis OQ Popula~ions of BFU-E From Bonc Marrow, Peripheral Blood a~ld Fetal Li~ er," ~Q~sl 74(1):49-5~, 1989; see also Iinch et al., ~Sh~dies of Circulanng Hemopoielic ProgeDilor Cells in Humall Fetal Blood,"
~9(":976-979, 1982). Selected culture conditions generally include growth ssandard cell a~ re media, without any other cytoldIles other than crythropoie~n. This preferentially allows fetal, but not maternal, nucleated e~hroid cells to grow. Parna21arly preferred media indudes Isc~ves' Modified Dulbecco's Medium (Glbco, Grand Island, N.Y.) coataining a final concentrauon of 20% fesal bo~e scn~m, and 2 U/ml pusified un~y kuman EPO.
Within the second method, fetal nuclea~ed, erythroid cells are preferen~ially enriched based upon their uptake of ammonium ions and the selecsive hemolysis of maternal blood cells, or more specifically, of maternal c~gthroid cells (see gcnerally Jacobs and Stewart, ''Ihe Role of Carbonic Anl~drase in Ccrtain Io~ic ExchaIlges Imrolving the Erythroc ~te,~ L Gen Phvsiol35 ~5~9-552, 1942; and Mares and Wiley, "Kine~ics of Car~o~ic A~hydrase in Wholc Red Cells as Mcasured by Transfer of Car~on Dio~cide aI~d Ammo~a,"
~:430440, 1970). Brie~y, the cells are i~cuba ed in the .. . .

, , :

2~ ~ 2`~3 wo 91/16452 PCr/US91/02789 prcsencc of ammoDia and c~londc ions and a Glrbonic anhydrase inhibitor under conditio~s and for a time sufficient tO allow acc~lmulanon of ammon~um ions within the cells. This geuerally takes approximately 5 to 30 minutes. Ihe cells are t~eQ irlcubated in t~e prcsence of ammo~ia aDd carbou dio~de such that selec~ve S hemolysis of matesnal blood cclls oc~s.
Within the context of the present i~venion, maly compounds may ~dc Q~itable sources of ammoma, chloride ions, a~d car~o~ dio~de. For e~ample, s~utable sources of ammonia include, among others, ammonia and the ammoDiUm salts. Suitablc sot~sccs of chloride ioDs i~clude, amoDg others, ~aa, 10 KCL Mga2, and CaC:12. Suitable sources for carbon d~oxide include, among others, carbon dio~ade in so~u~ion, carboDate, and bicarbonate. -Tn addi~ion, ma~y G~rbonic anhydrase in~ibitors are Imown in the ar~ inc~uding, among othcrs, most mono~alcnt aDions such as cyanidc and ~ate, monovalent sulfides, sul~onamides, and acctazolamide (see lindskog et aL ~.
15 E~= ~58 7, lg71; Ward and Cu~ 436, 19'72; and Poc3cer and Watamori, Bioche~L 12 '7475, 1g 73). Thc ca~onic a~ydrase i~l~itors s~ould be selec~ed so as to function under phgsiological corlditions.
Prcferrcd carbonic ar hydrase ir~ itors includc sulf~nilamide and acetazolamide.d cel~s may be released by vasious me~ods either subsequent 20 to, or prior to c~ichment. If bound cells are relcased pnor to emichment, they may ~c subse~uendy enriched using the above-described methods. ~arious meth~ds are known in the art for releasing cells. With n one such method, cells may bc csllt~red with or without gtokines. Cytokines, such as IL~ cause proliferation of ccl~s, or changes in the surfacc characteris~cs of the cells, such 25 that the cells or their progeny are released from a suppor~ Within another meehod, cleavage of the ligand or of the cell-ligasd bond may release the cell.
VaIious cleavable ligands and cleavi~g eDzymes are lmown in the ar~ includi~g among others, papaiII and ~ypsin~ Wit~ yc2 another method~ the cel3s may be released by mec~anical, gra~ntanorlaL or elec~romagne~ic forces. A particularly 30 prcfcrrcd mcthod is mcchaD;ical agitation, for example, by a~itatio~ of the beads through pipet~ng, stim~g, shaking, ~n~ration, or so~icanorL
Within another aspecs of the prese~t ir~ve~tioQ, a method is pro~vidcd whercin the cells are ~St cnriched, followcd by adsorption of fesal ~.
m~clea~ed, e~roid cel~s and remo~lal of unbound blood produas. Briefly, fetal 35 ccDs may be enriched by f~st ~cubanng maternal blood with ammo~ia and chloride ions, ~d a car~o~ic allhydrase inhibitor. Aftcr the cells have be~n inclbatcd undcr conditions and for a ~ne su~cient tO allow accumulauon of ;

2~)312~)r~
wo 91/16452 Pcr/US91/0278~

ammonium ions, the cel~s are trcatcd with ammonia a~d carbon dio~dc such tha~
sclecrive hcmolys,s of maternal blood cel~s occllrs. Fetal cclls may also be enriched by inalbat~on wi~h erythrop~icin as discussed above, or by a combined trea~ment using both esythropoietin and the method diso~ssed abo~e wherem ~he 5 cells are treated with ammonia a~ld carbon dia~de. Fur~emore, ~ tiscussed , the two methods may ~c pcrformcd in eithcr order.
T~e enricked cclls remaining a~er the method(s) descnbed above ma~ then bc ~mmob~izcd usng aI~y of the abovo disa~sscd me~hods, including:
(1) an immob~ized ligand which spccifically binds to ~ctal nucleatcd, e~2hroid 10 cclls remai~ing after eDrichment; (2) incubating cells remaming after enrichment unth a first member linkcd to a ligand, the ligand beirg capable of specificallybinding to fetal nucleated, erythroid cel~s, followed by adsorption of the celLs to a~
~mmobilized second mcmber, the sccond member being G~ablc of bindi~g to the Lsl member with an ~ffinity consta~t of greater than about 108~1; and LS (3) i~ ba~g cells rc~ a$cr cDric}~mcnt with a fi~t ligand, followed by incubation with a ~econd ligand which is chernically linked to a first membes which is capable of binding to the firs~ ligand under conditio~s and for a ~me sufficient to allow the seso~t liga~t to bind to the first ligand, followed by adsorption of the cdIs so an immobi3;zed sesond mcmbcr, the sesond member be~g capable of 20 bin~g to the fi~t member with an affini~y constant of greater than about 08 h~l, Subse~ue~ls so immobilizatiosl of the cells, u~ound blood products may be remo~ed USi~lg methods described above. If the cells were iDitially enriched using erythropoietin, subsequent to removi~lg unbou~d blood producss, 25 the cells may bc treated using the method wherei~ ammon-a a~d carborl dio~ade~re used. Similarly, if these cells were ~ally ~eated witS the method wherein ammoma and carbon dio~ade are used, subsequent to removing unbound blood produc~s, thc cells may be treated ~snth erythropoieti~
Fctal cel3s which have been e~siched ~om maternal blo~ld may be 30 c}larac~er~ed by i~cubaing 2he prefererltially erlIiched cells with a marlcercapable of selec~vely binding to fctal cells. A marker sclec~vely bmds to fe~al cells whe~ its presence on the target cells is greater than 1~fold more than theny on rnaIerrlal c lls. Vanous mar3cers are lmown in the art, including for ~am~le, an~odies to Alpha Fetal Protein (~ ) (see Kulozik ct aL, "Fetal

3~ Cclls irl thc Maternal CirQ~lation: Octection by Direct AFP-~no~luorescence," Human Gcnct. ~ 224, 1982), or annbodies tO antigen T (ue Y.W. Kan et al7 ~Conccntration of Fetal Red Blood Cells From a Mixrure , . ,: , , - ,:
:'' ' , . .

2 ~ 3 wo gl/16452 PCr/US91/02789 of Maternal and Fetal Blood by Anti-i Serum,~ 43(3):4111L5, 1974).
A~bodies tO the fetal cell mallccr may bc labcled using tcchni~ues well Imown m the art, and used to dctect t'ne presence of fetal cells.
Within a preferred embodi nent of the prescnt inve~on, ligand is S removcd from the cell surface t~ c methot tes~cd in a related a~plica~ion enti~ed "Mcthods for Rcmonng Iigands from a Par~clc Surface,"
ar.orney's doc~et m~mt.er 20007æ403, which is incorporatcd herein by refcrencc.
Such rcmo~ral may bc par~clllarly atvantageous pnor to cha~actenzatio~, as disalssed above.
As des.cnbed herein, enriched fetal cel~s have a ~aricr~r o~ uses. For example, through u~ siht hybridization the presencc of a seleaed genetic material may be deteacd in a fetal cell. Similarly, DNA or RNA amplification may 1so be used to descc~ a sele~ed gcnesic se~ence in fetal cells. The mcthods of the prescnt i~cntion are particularly usefill for fetal cclls which are cnnched tu such 15 an ~tent that they are ameDable to chromosomal typing by com~cn~onal cytogenesics techniqucs whcrein spreads of the cells' c~romoso~cs are examined undcr a microscope.
In si~u hybridization may bc us~d as a method for detec~ing the presence of a sclccted gcnetic material within cells (see Pinkel et aL, 1~oc. ?~Jatl.
20 ~ ~:913842, 1988; see also Hopma~ et al., ~Detc~ion of Numencal Chromosome Aberraions in Bladder Cancer by In S~n~ Hybn&~on.
Am J. o~ Path." t35(6):1105-1117, 1989). Brie~y, the gcnctic mau~ ssoc~ated with thc preferemially cDriched ce~ls is fi~st exposed usi~g tcchniques well known in the art. The genetic material is then inalbated wi1h a labeled probe capable of 25 specifically hybridizing to the genetic materiaL under condiions and for a time su~cient tO allow Iybndizatiosl to oca~r (see, for exa~nple, K E. Davies, Human Gene~ic Diseases. lRL Press, O~ord, U~, 1986). Finally, the prcsence of the h~oridized labelcd probc is deuctcd. Withi~ a preferred embodiment, the genetic material i . dena~rcd fter the step of exposirl~ Within ~he context of the present 30 i~e~non, genctic material includes whole chromosomes, D~TA, and RNA.
Various methods are also well known in the art for ampli~ng asld detecnng genetic materiaL For example, if present, a selectcd gcncnc scquence may be ~lificd us~g techni~ues well lmown ~ the art, and then probed for presencc of that se~ucncc (see Rogan et aL, ~An Improved Method For Prenasal 35 Diagnos~. of Gene~c Discases by A~alys~. of Amplified DNA Sequcnccs,~
ew EnP J o~le~ ~(16):985-990, 1987; sce also Wit~ and Eric3~so~L ~A ~apid Me2hod for Deucnon of Y-Chromosomal DNA from Dried Blood Spccimens by ,. . . .
, ,,. ~ :

. .
.

2~ 1203 wo sl/t64s2 PCr/US9~/0278 thc Polymerase CS~ caaion," ~ma~s~ 271-274, 1989). Methods for amplification indude Polymerasc ~n Reacnon (~C~") (see Mullis et al., U.S.
Patent No. 4,683,195; Mullis et aL U.S. Patent No. 4,683 ~02; and Mullis et al.,U.S. Patent No. 4,800,~59, w~ich are i~ porated here~n by refere&ce), and RNA-5 baced amplification tech~iques (see Iizardi et aL, Bio,~Tec}molo~ 1197-120Z, 1988; Kramer ct aL ~n~ 339:401~Q, 1989; and Lomcli Ct aL ~i~, S~i5~ ~(9):~ 83L 1989; see lco ~r~mer ct al, U.S. Patent No. 4,786,600, w~ is i~corpora~cd hese~ by rcfere~cc).
PCR is the most cornmorlly used method for ampli~ying DNA
10 sequences. Briefly, amplification enta~s adding the appropriate primer(s), en~nes and nucleo~des il~to a reac~ion rn~chre, followed by several (20-80) cycles of denaturation and a~nealing in order to amplif~ the small amou~t of targct DNA. ~he DNA mi~ure is thcn separated by electrophores~s a~d }~ybndized with a labeled probe to detect the presence of the target scquence of15 DNA.
l~e preferen~ally eDriched fetal cells may also be chrom~somally typed (see = =~ ~ D~ Rooney and B~ Czepulkousld (eds.), IRL
Press, Oxford, UX, 1986). Bne~ly, withirl a preferred cmbodimcnt, a sample contai~ing at least 1 fe~al cell in 105 o~cr cclls is al~red for 2~ days in Iscoves' 20 Modified Dulbecco's Medium (I~M) (Glbco, Grand Isla~d, N.Y.) contain~g a final conccn~adon of 20% fetal bovine serum (I;BS~ (HYCLONE~, Logan, Utah~ d 2 U/ml of hig~ly puri~ed recombinant esythropoienn (Terry Fox L;aboratory, Vancouver, B.C., Canada), in order to inease the proportioll of cells i~ metaphase. The cells are then incubated in colce~ud, fixed, attached tO25 microscope slides, trypsi~-treated, and stai~ed with Wnghrs staiIL The slides may then be sc~nned microscopically for abno~mal chromosomes.
lhe following examples are of ~ered by way of illustratioD, and ~lOt by way of ~imita~o~L

30EXAMP~ FS

~ . .
Carboxyla~on of a Polya~ylamide Gel 35Seventeen grams of dry Biogel P~O~, (5~100 mesh (wet), coarse bcads) (BIORAD, Catalog No. 15~1630, Richmond, Cali~) are addcd to 15 1 of 0.5 M NaHC03/0.5 M Na2C03. lhe pH is adjusted to 105 with ~aOH and 20~2~3 WO 91/16452 PCT/US91/0278g care~y stirred with a mLl~er (RZ~1, Carfamo, Wiarton~ Ontario, CaI~ada) so as not to damage the bcads for appro~mately 20 tO 30 minu~es. Ihe mL~ure is then placed in a 60C water bath After the mL~ure reaches a temperat~re of 60C, it is inQlbatcd for an additional 2 hours (at 60~C) w~th occasional stimng. T~e milccure 5 is then nmoved from the water bath, and p1aced in an ice bath to bring the mi~re temperat~lre down to room ~empc~ure.
Thc beads arc washed scveral ~ nth dis~lled or deior~ized ~vater, followcd by sc~veral washin~ of PBS ~ a coarse glass filter connected toa v~ m The G~oo~ylated gel may ~e stored in PBS at 4C, and is stable for up 10 to one ycar if ster~i~ed or stored with a preservative.

Example 2 Avidin Con~ugating the Carba~ylated Biogel PBS is fi~t removed irom a measurcd amount of carboxylated Biogel by filtenng with a coarse glass filter connec~ed to a vacuum. The gel is the~l equill~rated in dLt~led or deior~ized water for L~-30 rmmltes. Eq~ ration inwatcr cat~ses an a~pa~ion of the gel to a ~olume of about 4 times it's previously mcasured amounL lhc gel is resuspalded in 10 ml of disilled or deionized~vater 20 for each ml of gel (as ongin~lly measured ~ PBS).
Twe~ty $1g of 1~t~ 3~3 dimethylaminopropyl) carbo~de (El~C-HCl) (Sigma Chemical Co~ Catalog ~o. ET750, St. Louis, Mo.~ c added for each ml of gel as origi~ally measured. The pH is rapidly ad~usted to 55 by dropwise addition of Ha Care is taken to maintain the pH at 5~; pHs of less 25 thas~ 5.û or greater thaD 6.0 result in signifi~tly less ac~ivation of the Biogeh The mLsmre is s~ed for five minutes.
A~idin ~International Enzymes, Inc, Fallbrook Calif.) is dissolved at a conccntration of benvce~ 10 and 100 mg/ml in deionized water. Next, 500 ~g of a~idin is rapidly addcd for cach ml of gel (as origiually mcasured in PBS). l~e 30 m~rc is ~ red for 15 hours. Next, 2 M glycine is added to gnre a final co~centraion of 0 ~ M glycis~e i~ the mL~ re, asld stirred for an additional 1 hour.
Thc gel is washed with several ~olumes of PBS using a coa~se glass filtcr and v~m, a~d stored in PBS with 0.1% NaN3 at 4cC The gel is stable for appr =cly onc ycar.
E3am~~
~smunoadsorp~ao~ of Matcr~al Cells WI~ Thc Two-Step Mcthod 2~81'~3 wo g~ 52 Pcr/uS91/02789 PRePAR~nON 0~ C~
Twe~t:y milliliters of blood i5 ob~uncd ~om a pregnaIlt female a d suspendcd in an equal volume of PBS with 1~o Bavinc Scrum Album~ (BSA) S (Sigma Chemical Co~ St. Louis, Mo.), in four 50 ml centrifuge tubes. Thc number of nucleatcd cclls/ml is counted iII the follow~g manner: A 50 l~l aliquot of mLsed, a~coagulatcd whole blood is diluted into 3 ml of a 3% acetic acid solurion. Aftcr vo~ ~ pl samples of dilutcd blood are loaded into cach of two cham~ers of a hemacytometcr. After about 3 mimltes to allow settling of the 10 cells, the nuclei of cells, which are al~nost all lysed by the acc~c acid, are counted in the four ruled fields per chamber of a hemacytometer wi~h improYed ~Jeubauer ruli~gs (VW~ Sacntific~ San Fra~cisco, Calif ), each field represe~g 0.1 x 10-3 ~I sa~le volume. The average number of ~uclei per field is multiplied ~y a dilutiorl faaor of 61 x 104 to calallate the ~umber of nucleated cells per milliliter 15 of whole blood If thc a~ erage is lcss than 10 Iluclci p~r field, the procedure is re~ted except 50 al bl~d is d~utcd into orlly 1 ml acetic acid, with a resultingncw dilution factor of 21 x 1~4.
Each tube of dilused blood is m~derlaycrcd w~ S ml of Histopa~ue 1077 (Sigma Chemical Co.) and c~nt~ed at 700 g for L5 m~utes at room 20 tempera~re. Cel~ at the illterface are colle~ed a~ld washed once in PBS plus 1%
BSA. The pellet is resuspended i~ 100 ~Ll in PBS plus 1% BSA.
Twenty micrograms/mil~iIisers of anh~ erml receptor an~body (Becton Dicki~son, Irnmunocytome~ry Systems, Mountain View, Ca~if.) is added to the mLsmre a~d incubated for 15 minutes orl iee. The cells are then washed 25 oIlce~ith 4 ml of PBS plus 1% BSA and c~;fuged at approDma~ely 400 g for 5 minutes.
The cells are then gently resuspended to 1 ml and 1 ~g/ml of bio~ylated goat anti-mouse IgG (Sou~ern Biotech, Bi~ am, Ala) is added.
Ihe mixrure is incllbated for 15 minutes on ice and washed twice as described 30 above with PBS plus 1% BS~

PREP~AnON OF COLLnUNS
Carboxylated Biogel P-30~ (prepared as described above) is allowed to equill~rate to room tempcrature and placed in a K9/L5 column (Pharmacia, 35 Piscataway, NJ.) to a total bcd hcight of 1 cm. The column is washed with PP~s~
followcd by washes with PBS plus 5% BSA This colum~ func~ions as a ~prc-columr~" I'he a~ndin column corltains avidin conjugatcd ~iogel PbO~ which LS

.. - . ~ ... , .-.. .

:.

2 ~
wo 9l/l6452 PCr/US91/02789 prep~ed as descnbed above. The a~dirl~o~jugated Biogel is allowed to equil~rate to room temperature, and piaced iD a K9/15 column to a total bed hcight of 4 cm. The column is thcn washed with sevcral volumes of PBS, followed by washes with PBS pllL 5% BSA.

INMUNa~DSORrS~ON OP C~FT T C
Cells which have bcen prcpared as des~ed abave are resus;pended i~ PBS plus 5% BSA to a volume of 1 mL The cclls are then gently transferred onto the top of thc gcl bed of thc pre~olum~ filter. lhe cells arc allowed ~o fi~tes 10 through thc pre-column and are wa_hed with 1 ml PBS plus 5~ BS~ A
penstaltic pump (Cole-Parmer, Roc~ord, Ill.) controls flow from the avidi column to a rate of about 1 ml/minute. Once the cells ha e almost run down to thc top of ~e a~ idin column bed, 1-2 rnl of PBS plus 5% BSA is added to the topof the avidin column in order to wash out remaiIIing cells. The colum~ is w~ched15 out ~nth 4-6 ml af PBS plus 5% BSA, followed by 4-6 ml of PBS.

R~MOVAL O~ SOR~ED C~5 F~OM TH~A~Dn~l COLUMN
The andin column is placed on top of a 15 ml c~ntrifuge tube. The ~alvc of the calumn is opened and 15 ml af ~P~ 1 is added to the col~mn w~th a 20 ~ide bore, 9-inch ~ansfcr pipette. The RPM~ ~ added to the column wh~le the pipe~e is used for mechanically agita~g alld resuspending the cell bed, ~u_ allowing ce~ls to become detached from the gel matnx, and to filter into the centrifilge tube. The tube i_ then centrifi~ged at 400 g for 5 minutes and resuspended in ccll c~llture media as descnbed below.

ExasnD~e 4 Prefercn~ ichment of Fcsal ~ucleated, E~roid Cells E.~sr o~ FErAL CE~S WrrH ER~oPon~r~
Cells which are separated from the A~ Biogel column above, are rcsuspended in Iscoves' Modiiied Dulbco's Medium (I~M) (Gibco, Gra~d Island, N.Y.) containing a fi~al concentration of 2~% fetal bovine scrum (FBS) (~IYCLONE~, Log;m, ULah), a~d 2 U/ml of hig~ly purified recomb nant esy~ropoicin (Ter~y Fox I~boraLory, Vana)uver, B.C., Callada). Cclls are dilutcd to 5 x 106 lucleated c 31s/ml and 200 ~1 LS platcd into cach wel~ of a 96 wcll ~ssue cul~re plate with round bo~oms (Corning Glass Works, Coriing, N.Y.).

. ~ . .
' wo 91/16~s2 2 3 ~ 1 2 ~ 3 PCr/US91/0278~

Enrichrnent of Fetal Ce11s by Ammoniurn Ion Differen~iauon Cells which arc separated ~om the Avidin column above, are adiusted to a conccsltration of less than 2 X 10~ nucl~-qted cclls/mL One volurne S of tbc cell mi~ure is cl~illcd, and a solu~on a~ 29C containing 18 ~olumes of0.1844 M NH4a ~d 2 volumes o~ 10 aM acetazolamidc is addcd. After 2 m~utes, 2 vohlmes of 3mM NH4HCO3 is ra~idly added, and the whole mL~ure gen~r stirrcd for 3 mi~t~ Cclls are was~c~ se~eral t~nes with PBS by ccntrifugaio~ in order to removc ccllular de~ris.
am~le 5 In Si~ Hybridization Enric~ed cclls arc exposed to a 12ypoto~ic solution (.~75 M KCl) for LS 1~ minutes at 3'PC The tubes are i~vertcd once during thc i~albation to kccp dle cclls suspcntet. Twenty drops of fres~ly prcpared f~a~ve (3:1 mctha~ol:acetic acid) is added to the cells, vorte~ed9 a~d then cc~ifilged for 8 mim~tes at appmnamtely 250 g. ~rcsh fixative is added to the cells, followed by incubanon for one hour at room tcmperature. The cells are centrifuged for 8 minutes at 20 appr~tely 250 g. Frcsh fDca~c is added and the process is repcated one more time. Fma~ly, the cells are resuspc~ded in a small amoT~t of fi~a~e and placed at 4C overDig~L
The ~ext day the cells are vortexed and placed onto microscope s~ides (Baxter, McGaw Par~c, ~) whic~ have been cleaned with ethanol and 25 dipped ihl dis~lled water. The s1ides are allowed to dry for two days at room temperature. .
The slides are healed in 70% ~ormamide in 2x SCC (030 M NaCl, Q030 M Na ~trate) to 68C-70C for æ5 mi~utes. The slides are then immediately placed in a rinse of 7û% ethanol in waur. Following the ri~se, the slides are 30 placed successrve~y in 7a~a, 95~0, aIld 100~ e~a~ol solu~o~s for 5 ~tes each Each alcohol solution must be ~tained at ~ -20C The s~ides are air d~ied.
A probe is prep~ed from plasmid DNA accordi~g to thc me~hod of Page et al (~Singlc coy scquencc hybridizcs to polymorphic a~d homologous loci on human X alld Y chromosomes" ~12:~352-5356, 1982), from Amcrican 35 Type Cul~c Collecnon (ATCC) ~o 57261; exccpt that biotin dATP is ~corporatcd into t~e probe. Twenty ~ve microlitcss of ~e bio~ir,rlated probe (S
~g/ml) pcr slide is placed in a mi~ofuge tube. The probe is hcated to 70C for S

' `- , , "'. : ~' ' 2~3~7~
wo gl/16452 Pcr/uss1/o2789 mimltes thcn immediatcly placcd on icc. Twcnty microliters of the probc soluion is placed onto each slide a~d covered with a Z x 40 mm covesslip. The slides ~replaced ~to a box with a WCt papcr toweling li~er and ~cubatcd at 37~C for 12-18 houIs.
A 50% solution of fo~de in 1 x SCC LS wa~cd to 3rC
Covcrslips are rcmo~rcd from the slides and immes~ed into the 50% formamide solution for 30 m~utes. Thc slides arc theu placed ~ 2 ~ SCC solution for 30 ~tcs ~ith gentlc roc3~ng, thcn in 1~ SCC for 30 ~tes ~mh gesltle ~g.
Fluoresceinated a~ridi~ (Vector, Burlingame, Calif ) is dilutcd 1:1000 (1 5~glml).
The bac3c of the slidc and around cell area are wiped. Two hundred microliters of the avid~-fluorescein is added to each slide alld incubated i~l thc ~ox for 30 minutes at room tcmperature~ Thc slides are rinsed seq~entially in 1) 4 x SCC for 10 m~utes ~i~ rocl~g, 2) 4 ~ SCC, 0.1% Twee~-20 for 10 m~nutcs without roc3~ing, 3) 4 x SSC for 10 minstes with roc~ The bac3~ of the slide and arou~d 15 ccll arca is wipcd of. Te~ microlitcrs of anti-f~de p~us propidium iodi~G (10 ml PBS7 100 mg p-PheD5~1ene diaminc, 90 ml glyceroL pH 8.0, 10 ~g/ml propidium iodide) is added. The slides are covered with caverslips and stored at 4C The slides may be stored for scveral days if nesessary. Target DNA ma~r bc obses~ed u~der a microscopc by the presence of fluoresceuce.~
am~le 6 Chromosomal Typi~g The e~rsched cells are exposed to 1 I~g of colcemid (Sigma, 25 St. Louis, Mo.) for one hour at 37C A }IypotoDic solunon (0.075 M Ka) is added to the cells and incu~ated for 12 miIlutes at 3TC. The tubes are i~Iverted once dunng the incuba~aon to keep the cells suspended. Twenty drops of freshly prcpared fixat*e (3:1 mcthanol:ace~c a~d) is added to the cdls, vorte~ccd, and then cent~gcd for 8 mi~lutes at appm~mately ~50 g. Frcsh fi~a~n~e is added to 30 the cells, followet by incuba~ion for 1 hour at room tempe~a~ure. T~e ccl~s are centrifilged for 8 minutes at ~pro~omately 250 g and ~esh ~ve is added a~
This process is repealed one morc time. Finally, the cells are resuspended in a small arnou~lt of fixa~ve and placed at 4C overmg~t. Thc nest day the ccLls arevo~exed and piaced onto micros~pe slidcs (Ba~ctcr, McGaw Par~, ~L) which have 35 been cleaned wish esha~ol and dipped 3n disdlled water. The slides are allowed to itr dry for about 34 dag~, and then are treated with Q005% tlypsill ~Difco Bactotsypsin, VWR Scicntific, San Franc2sco, Cali) for 30 to 35 seconds. The .

20~120~ `
wo 9l/l6452 PCr/US91/02789 slides are washed nlnc PBS Plu~ 1% FBS, followed by washing iIl PBS o~ly.
l~e cclls arc stainet wi~ Wright's solu~on (Si~ma Chemical Co., St. Louis, Mo.),fo~lowcd by rwo washes w~th tcionized water. The s3ides are scanned for e~vidence of mc~haso cçl~s and ~rped by 0nvcntio~al cy~ga~èti.
S ~rom the forcgoi~g, it wal be apprccia:tcd t~, alehough specific cmbodimc~ of ~e i~Ive~ion h~nrc ~een dcs~bcd herein for the pu~poses of m~on, ~arious modifica~ions m;ry be madc withont devia~g ~om the Spiril and scope of the imre~l~oD. ~ccordingk~r, the m rcn~ion is t ~nited e~cept as by t~c a~pended da~s.

Claims (72)

Claims

1. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated. erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) removing unbound blood products; and (c) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

2. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) removing unbound blood products;
(c) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions ant for a time sufficient to allow accumulation of ammonium ions within the bound cells; and (d) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.

3. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) removing unbound blood products;
(c) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells;
(d) incubating the cells with accumulated ammonia ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs; and (e) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

4. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of the maternal blood with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within said cells;
(b) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs;
(c) incubating cells remaining after hemolysis with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells; and (d) removing unbound blood products.

5. The method of claim 4, including, subsequent to removing said unbound blood products, incubating the cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

6. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of the maternal blood in the presence of erythropoietin such that the fetal cells are enriched;
(b) incubating the enriched cells with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells; and (c) removing unbound blood products.

7. The method of claim 6, including, subsequent to removing said unbound blood products, incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells; and incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.

8. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood in the presence of erythropoietin such that the fetal cells are enriched;
(b) incubating the erythropoietin enriched cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within said cells;
(c) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs;
(d) incubating the enriched cells from step (c) with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells; and (e) removing unbound blood products.

9. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within said cells;
(b) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs;
(c) incubating the enriched cells from step (b) in the presence of erythropoietin such that the fetal cells are enriched;
(d) incubating the erythropoietin enriched cells with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and far a time sufficient to allow specific binding of said ligand to the cells; and (c) removing unbound blood products.

10. The method of claims 1 through 9 wherein said ligand is selected from the group consisting essentially of antibodies, erythropoietin, and transferrin.

11. The method of claims 1 through 9, including, subsequent to the step of removing said unbound blood products, releasing the immobilized cells.

12. The method of claim 11 wherein said cells are released by mechanical agitation.

13. The method of claims 1 through 9 wherein the step of removing comprises rinsing the bound cells with a physiological solution.

14. The method of claim 13 wherein said physiological solution is selected from the group consisting of PBS, PBS plus albumin, normal saline, and cell culture media

15. The method of claims 1 through 9, including, subsequent to the step of preferentially enriching the fetal cells, incubating the enriched cells with a marker capable of selectively binding to said fetal cells.

16. The method of claims 1 through 9 wherein said ligand is immobilized on a solid support selected from the group consisting of hollow fiber, beads, magnetic beads, plates, dishes, flask, meshes, screens, solid fibers, membranes, and dipsticks.

17. The method of claims 2, 3, 4, 7, 8, or 9 wherein said carbonic anhydrase inhibitor is selected from the group consisting of sulfanilamide and acetazolamide.

18. The method of claims 2, 3, 4, 7, 8, or 9 wherein said chloride ions are selected from the group consisting of NaCl, KCl, MgCl2, and CaCl2.

19. The method of claims 2, 3, 4, 7, 8, or 9 wherein said ammonia is selected from the group consisting of NH4Cl and ammonium salts.

20. The method of claims 2, 3, 4, 7, 8, or 9 wherein said carbon dioxide is selected from a group consisting of carbon dioxide in solution, carbonate, and bicarbonate.

21. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with a first member chemically linked to a ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1;
(c) removing unbound blood products; and (d) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

22. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with a first member chemically linked to a ligand capable of specifically binding to said cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1;
(c) removing unbound blood products;
(d) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells; and (e) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.

23. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with a first member chemically linked to a ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1;
(c) removing unbound blood products;

WO 91/16452 PCT/US9l/02789 (d) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells;
(e) incubating the cells containing accumulated ammonium ions in the presence of ammonia ions and carbon dioxide such that selective hemolysis of maternal blood cells occurs; and (f) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

24. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of the maternal blood with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within said cells;
(b) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs;
(c) incubating cells remaining after hemolysis with a first member chemically linked to a ligand capable of specifically binding to said cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(d) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1; and (e) removing unbound blood products.

25. The method of claim 24, including, subsequent to removing said unbound blood products, incubating the cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

26. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of the maternal blood in the presence of erythropoietin such that the fetal cells are enriched;
(b) incubating the enriched cells with a first member chemically linked to a ligand capable of specifically binding to said cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;

(c) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1; and (d) removing unbound blood products.

27. The method of claim 26, including, subsequent to removing said unbound blood products, incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions dons and for a time sufficient to allow accumulation of ammonium ions within the bound cells; and incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.

28. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood in the presence of erythropoietin such that the fetal cells are enriched;
(b) incubating the erythropoietin enriched cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within said cells;
(c) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs;
(d) incubating the enriched cells from step (c) with a first member chemically linked to a ligand capable of specifically binding to said cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(e) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1; and (f) removing unbound blood products.

29. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within said cells;

WO 91/16452 PCT/US91/0278?

(b) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs;
(c) incubating the enriched cells from step (b) in the presence of erythropoietin such that the fetal cells are enriched;
(d) incubating erythropoietin enriched cells with a first member chemically linked to a ligand capable of specifically binding to sad cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(c) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1; and (f) removing unbound blood products.

30. The method of claims 21 through 29 wherein said ligand is selected from the group consisting essentially of antibodies, erythropoietin, and transferrin.

31. The method of claims 21 through 29, including, subsequent to the step of removing said unbound blood products, releasing the immobilized cells.

32. The method of claim 31 wherein said cells are released by mechanical agitation.

33. The method of claims 21 through 29 wherein the step of removing comprises rinsing the bound cells with a physiological solution.

34. The method of claim 33 wherein said physiological solution is selected from the group consisting of PBS, PBS plus albumin, normal saline, and cell culture media.

35. The method of claims 21 through 29, including, subsequent to the step of preferentially enriching the fetal cells, incubating the enriched cells with a marker capable of selectively binding to said fetal cells.

36. The method of claims 21 through 29 wherein said second member is immobilized on a solid support selected from the group consisting of hollow fibers, beads, magnetic beads, plates, dishes, flasks, meshes, screens, solid fibers, membranes, and dipsticks.

37. The method of claims 22, 23, 24, 27, 28, or 29 wherein said carbonic anhydrase inhibitor is selected from the group consisting of sulfanilamide and acetazolamide.

38. The method of claims 22, 23, 24, 27, 28, or 29 wherein said chloride ions are selected from the group consisting of NaCL, KCl, MgCl2, and CaCl2.

39. The method of claims 22, 23, 24, 27, 28, or 29 wherein said ammonia is selected from the group consisting of NH4Cl and ammonium salts.

40. The method of claims 22, 23, 24, 27, 28, or 29 wherein said carbon dioxide is selected from a group consisting of carbon dioxide in solution, carbonate, and bicarbonate.

41. The method of claims 21 through 29 wherein the first member-second member binding pair are selected from the group consisting of biotin-avidin, biotin-streptavidin, biocytin-avidin, biocytin-streptavidin, methotrexate-dihydrofolate reductase, 5-fluorouracil-thimydylate synthetase, and riboflavin-riboflavin binding protein.

42. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with a first ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said first ligand to the cells;
(b) incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand;
(c) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1;
(d) removing unbound blood products; and (e) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

43. A method for enriching fetal nucleated, erythroid cells from maternal blood comprising:
(a) incubating a sample of maternal blood with a first ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said first ligand to the cells;
(b) incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand;
(c) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1;
(d) removing the blood products;
(e) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allowaccumulation of ammonium ions within the bound cells; and (f) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternalblood cells occurs.

44. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with a first ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said first ligand to the cells;
(b) incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand;
(c) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1;
(d) removing unbound blood products;
(e) incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allowaccumulation of ammonium ions within the bound cells;

(f) incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of other than fetal cells occurs; and (g) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

45. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of the maternal blood with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a timesufficient to allow accumulation of ammonium ions within said cells;
(b) incubating the cells containing accumulated ammonium in the presence of ammonia and carbon dioxide such that selective hemolysis of maternalblood cells occurs;
(c) incubating the cells remaining after hemolysis with a first ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said first ligand to the cells;
(d) incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand;
(e) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1; and (f) removing the blood products such that fetal cells are enriched.

46. The method of claim 45, including, subsequent to removing said unbound blood products, incubating the cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

47. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of the maternal blood in the presence of erythropoietin such that the fetal cells are enriched;
(b) incubating the enriched cells with a first ligand capable of specifically binding to said cells under conditions and for a time sufficient to allow binding of said first ligand to the fetal nucleated, erythroid cells;

(c) incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand;
(d) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1; and (e) removing unbound blood products.

48. The method of claim 47, including, subsequent to removing said unbound blood products, incubating the bound cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the bound cells; and incubating the cells containing accumulated ammonium ions in the presence of ammonia and carbon dioxide such that selective hemolysis of maternalblood cells occurs.

49. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood in the presence of erythropoietin such that the fetal cells are enriched;
(b) incubating a sample of blood with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within said cells;
(c) incubating the enriched cells with a first ligand capable of specifically binding to said cells under conditions and for a time sufficient to allow specific binding of said first ligand to the cells;
(d) incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to the first ligand;
(e) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1; and (f) removing unbound blood products.

50. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:

(a) incubating a sample of maternal blood with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within said cells;
(b) incubating the enriched cells with a first ligand capable of specifically binding to said cells under conditions and for a time sufficient to allow specific binding of said first ligand to the cells;
(c) incubating the enriched cells from step (b) in the presence of erythropoietin such that the fetal cells are enriched;
(d) incubating the sample with a first member chemically linked to a second ligand capable of specifically binding to the first ligand under conditions and for a time sufficient to allow the second ligand to bind to a first ligand;
(e) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1; and (f) removing unbound blood products.

51. The method of claims 42 through 50 wherein said ligand is selected from the group consisting essentially of antibodies, erythropoietin, and transferrin.

52. The method of claims 42 through 50, including subsequent to the step of removing said unbound blood products, releasing the immobilized cells.

53. The method of claim 52 wherein said cells are released by mechanical agitation.

54. The method of claims 42 through 50 wherein the step of removing comprises rinsing the bound cells with a physiological solution.

55. The method of claim 54 wherein said physiological solution is selected from the group consisting of PBS, PBS plus albumin, normal saline, and cell culture media.

56. The method of claims 42 through 50, including, subsequent to the step of preferentially enriching the fetal cells, incubating the enriched cells with a marker capable of selectively binding to said fetal cells.

57. The method of claims 42 through 50 wherein said second member is immobilized on a solid support selected from the group consisting of hollow fibers, beads, magnetic beads, plates, dishes, flasks, meshes, screens, solid fibers, membranes, and dipsticks.

58. The method of claims 43, 44, 45, 48, 49, or 50 wherein said carbonic anhydrase inhibitor is selected from the group consisting of sulfanilamide and acetazolamide.

59. The method of claims 43, 44, 45, 48, 49, or 50 wherein said chloride ions are selected from the group consisting of NaCl, KCl, MgCl2, and CaCl2.

60. The method of claims 43, 44, 45, 48, 49, or 50 wherein said ammonia is selected from the group consisting of NH4Cl and ammonium salts.

61. The method of claims 43, 44, 45, 48, 49, or 50 wherein said carbon dioxide is selected from a group consisting of carbon dioxide in solution, carbonate, and bicarbonate.

62. The method of claims 42 through 50 wherein the first member-second member binding pairs are selected from the group consisting of biotin-avidin, biotin-streptavidin, biocytin-avidin, biocytin-streptavidin, methotrexate dihydrofolate reductase, 5-fluorouracil-thimydylate synthetase, and, riboflavin-riboflavin binding protein.

63. The method of claims 42 through 50 wherein said second ligand is an antibody.

64. A method for enriching fetal nucleated, erythroid cells from maternal blood, comprising:
(a) incubating a sample of maternal blood with biotinylated anti-erythroblast antibody under conditions and for a time sufficient to allow the antibody to bind to the fetal nucleated, erythroid cells;
(b) passing the cells through a packed bed of porous material having avidin bound to the surface thereof so as to adsorb the biotinylated antibody to the avidin on the surface of the porous material, thereby binding the fetal cells and adult cells to the porous material;

(c) rinsing the packed bed to remove unbound blood products;
(d) releasing bound cells by mechanical agitation of the packed bed;
and (e) incubating the released cells in the presence of erythropoietin such that the fetal cells are preferentially enriched.

65. A method for enriching fetal nucleated, erythroid cells from maternal blood comprising:
(a) incubating a sample of maternal blood with biotinylated anti-erythroblast antibody under conditions and for a time sufficient to allow the antibody to bind to the fetal nucleated, erythroid cells;
(b) passing the cells through a packed bed of porous material having avidin bound to the surface thereof so as to adsorb the biotinylated antibody to the avidin on the surface of the porous material thereby binding the fetal cells and adult cells to the porous material;
(c) rinsing the packed bed to remove unbound blood products;
(d) releasing bound cells by mechanical agitation of the packed bed;
(e) incubating the released cells with ammonia and chloride ions and a carbonic anhydrase inhibitor under conditions and for a time sufficient to allow accumulation of ammonium ions within the released cells; and (f) incubating the released cells in the presence of ammonia and carbon dioxide such that selective hemolysis of maternal blood cells occurs.

66. A method for enriching fetal nucleated, erythroid cells from maternal blood, and for detecting the presence of a selected genetic material comprising:
(a) incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) removing unbound blood products;
(c) incubating the bound cells in the presence of eryhropoietin such that the fetal cells are preferentially enriched;
(d) exposing the genetic material associated with said preferentially enriched cells;
(e) incubating the genetic material associated with the fetal cells with a labelled probe capable of specifically hybridizing to said genetic material under conditions and for a time sufficient to allow hybridization to occur; and (f) detecting the presence of the hybridized labelled probe.

67. The method of claim 66, including, subsequent to the step of exposing the genetic material, denaturing the genetic material.

68. A method for enriching fetal nucleated, erythroid cells from maternal blood, and for typing chromosomes from said cells comprising:
(a) incubating a sample of maternal blood with an immobilized ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) removing unbound blood products;
(c) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched; and (d) typing the chromosomes associated with the enriched fetal cells.

69. A method for enriching fetal nucleated, erythroid cells member maternal blood, and for detecting the presence of a selected genetic material comprising:
(a) incubating a sample of maternal blood with a first member chemically linked to a ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1;
(c) removing unbound blood products;
(d) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched;
(e) exposing the genetic material associated with said preferentially enriched cells;
(f) incubating the genetic material associated with the fetal cells with a labelled probe capable of specifically hybridizing to said genetic material under conditions and for a time sufficient to allow hybridization to occur; and (g) detecting the presence of the hybridized labelled probe.

70. The method of claim 69, including, subsequent to the step of exposing the genetic material, denaturing the genetic material.

71. A method for enriching fetal nucleated, erythroid cells from maternal blood, and for typing chromosomes from said cells comprising:
(a) incubating a sample of maternal blood with a first member chemically linked to a ligand capable of specifically binding to fetal nucleated, erythroid cells under conditions and for a time sufficient to allow specific binding of said ligand to the cells;
(b) adsorbing the cells to an immobilized second member, said second member capable of binding to the first member with an affinity constant of greater than about 108 M-1;
(c) removing unbound blood products;
(d) incubating the bound cells in the presence of erythropoietin such that the fetal cells are preferentially enriched; and (e) typing the chromosomes associated with the enriched fetal cells.

72. A method for typing chromosomes of fetal nucleated erythroid cells, comprising:
(a) incubating the fetal nucleated erythroid cells in a media containing erythropoietin under conditions and for a time sufficient to induce metaphase in said cells;
(b) fixing the DNA of said cells;
(c) staining the fixed DNA such that chromosomes may be observed;
and (d) examining the stained DNA thereby allowing the typing of the chromosomes.