Note: Descriptions are shown in the official language in which they were submitted.
2 1 2 2 1 3 8 Pcr/uss2/o9o24
. " 1
A MEYHOD FO:R ~iNl~G F~ETAL PROGENITOR OELLS
S F~OM ~TERNAL BLOOD
~ss Refer~nce to Rel~ç~li~
This application is a continuation^in-part of pending lLJ.S.
10 Application Serial No. 07/513,057, ~iled April 23, 1990.
Technical Field
The invention relates to a method of enriching ~etal progenitor cells
from maternal blood.
~a~kgr~und ~the ~nventi~n
~ ppro~imately 5.3 million women become pregl~ant in the IJnited
$tates yearly, resulting in 3~8 million del*eries. There are an additional 10 million
deliveries in the other af~uent countnes i~ the world. Pre~atal testiIlg is used Ln a
~: 20 subgroup of these women who have a si~ficant risk of ha~ing a fetus with a
genetic disorder such as Dowll's Syndrome.
At present, tlhe only way of diagnosirlg fetal disorders is to obtain
cells from~ the amniotic iluid (amniocentesis) or the surface of th.e ~etal sac
c~orionicvillus) of the mother. Th-~se procedures are exper~iYe and carry a risk; ~ ; 25 of s~ontaIleous abortion of between Y2% to 1%. Because ~ the risk of
spontaneQus abor~ioIl, these collection procedures are ~eco~nended only for
women who are at a high-~isk of carrying a child with a genetic defect. For
example, only women o~er age 35 are advîsed to have the test because the risk ofDown's SyIldrome is higher in that group. Some of these women refuse ~he test
3û beeause of the risk of spontaIleous abortîon. Even though many of these high-nsk
women are tested, only a i~action of fetuses with Down's Syndrome are de~ec~ed.
The hîgh-risk women represent such a small po~ion of the women having chîldren
that the low-risk population stîll delîvers most of the afflic~ed children. Eîghty
percent of those chîldren born wieh Down's Syndrome are from the "l~w-risk,"
35 under 35-year-old group~ This situation is also true of many other genetic de~ects
or disorders.
WO 93~08269 2 1 2 ~ 1 ~ 8 PC~r/US92/09024
I~ is therefore desirable to provide a test that wnuld resolve this
testing dilemma by providi~g a safe method which could be available to all
pregnant women, irrespective of the risk factor aIld without nsk of spon~eous
abortion. Although it is kn~ that fe~al cells c~rculate ill t~e blood stream of
5 pregnant women ~see Kulozik and Pawlow~tzl~, "Fetal C,'ells in the Maternal
Circula~on: Detection by Direct AF~-Immunofluorescence," Human Genetics
62:221-224 (198~)), fetal cells ~re presen~ in such low concentrations that the
procedures necessary to isolate them are extremely difficult and time-consuming;For example, ~erzenberg ~t a~, "Fetal Cells in the Blood of Pregnant Women:
10 Detection and Enric~ment by F~uorescence-Activated Cell Sorting," Proc. Nat~
Aca~ Sci.9 USA 76:1453-1455 (1979), used a Fluorescence-Activated Cell Sorter
(FACS) in order to detect fetal cells in maternal blood. The procedure used,
however, is not adaptable to routine clinical testing procedures, in part, due to the
great expense and expertise required to run a FACS machine. The method of
15 Hertzenberg et a~ is also defic~ent ~or routine clinical testing bec~use it requires
determinatioll of ~ ~pes. Bianchi et a~ "Direct Hybndization to DNA From
Small Numbers of Flow-Sorted Nucleated Newbom Cells," Cytometry 8:197-202
(1987~ also used a FACS machine to detect nucleated cells, althvugb the blood
sample was not obtai~ed ~om the mother but from the newborn's umbilical cord.
Tbe prese~t invention provides a metbod for enriching fetal cglls
that ~vercomes these disadvantages, and furtber provides other related
advalltages.
Sl~mmaly ~f ~e Invention
: 25 T~e preseIlt inYen~ion is also directed ~oward methods for enriching
feta~ nudeated~ e~hroid cells from maternal blood. Within one aspect of the
present inven~ion, such a metXod compAses the s~eps of (a) incuba~dng a sample of
matemal blood with an immobilized ligand capable of specifically bi~ding ~o fetal
~, nucleated, e~hroid cells lmder conditions and for a time suf~c~ent to allow
30 specific bindillg of the ligalld to the cells, (b~ remo~g unbound blood products,
d (c) ~cubating the bouIId cells in the presence of erythropoietin such that thefetal cells are prefere~ially enrichçd. Withill orle embodiment of this aspect of
the inventioD, the immobilized ligand is an immobili~ed antibody.
Within another aspect of the invention, a method is pr~vided for
3~ enriching ~etal nucleated, erythroid cells from maternal blood, compnsing thesteps of: in~bating a sample of maternal blood with a first nnember chemically
li~ced to a ligand capable of speci~ically binding ~o fetal nucleated, e~ythroid cells
WO ~3/08269 2 ~ Pcr/uss2/o~û24
under conditions and for a time sufficient to allow specific binding of the ligand to
the cells; adsorbing the cells to an immobilized second member, tbe second
member beL11g capable of binding to the first member with an affinit~ constant of
greater thaIl about 108 M-l; remo~ing unbound blood produc~s; and incubating
5 the bound cells ~n the presence of erythropoietin such that the fetal cells are
preferentially enriched. Suitable first member~second member binding pairs
include biotin-~vidin~ biotin-streptavidin, biocytin-avidin, bio~rtin-streptavidin,
~:methotrexate-dihydrofolate reductase, 5-fluorouracil-thimydylate synthetase, and
riboflavin-riboflavin binding protein. Within one embodiment of this aspect of the
10 invention, the first member which is chemically linked to a ligand is a biotinylated
antibody and the i~obilized second member is immobilized avidin.
~:Within another aspect of the present invention, the method
comprises the steps of: ~ncubating a sample of rnaternal blood with a first ligand
capable of specifically binding to fetal nucleated, eIythroid cells under conditions
15 a~d for a time suffi~ient to allow specific binding of the first ligand to the cells,
incubating the sample with a first member chemically linked to a second ligand
~;capable of specifically bin~g to the first ligand under conditions and for a time
suf~cient to allow the second liga~d:to bind to the first liga~d; adsorbing the cells
to aII immobilized second member, the second member capable of binding to the
20 first member with an afEinity coIlstant of greater than about 108 M 1; removing
unbound blood~ products; and incubating the bound cells in the presence of
erythropoietin such that the fetal cells a~e pre~erentially enriched. Within oneembodiment, the first Iigand is an antibody which specifically binds to fetal
nucleated, e~roid cdls. Within preferred embodiments, the first member which
25: is chemically linked to the second ligand is a biotinylated antibody. In such an
embod~ent, the immobiliæd second member is immobilized avidin.
As an alternative to the step o~ incubating bound cells in the
:~ presence of e~thropoietin, another aspect of the present invention comprises the
steps of: (a) incubating the bound cells wi~h ammonia and chloride ions and a
3~ ca~bonic anhydrase i~ibitor under conditions aIId for a time sufficient to allow
accumulation of a~nonium ions within the bound cells, and (b) incubating the
bound cells containing accumulated ~nmonium ions in the presence o~ ammonia
~:and c~bon dioxide such that selective hemolysis of maternal blood cells occurs.
I~ addition, the present invention may also be perfo~ned usillg a combination of35 both erythropoietin enrichment and the method described above wherein the cells
are incubated with ammoI~ia and chloride ions and a carbonic anhydrase inhibitoruIlder conditions and for a time ~u~Scient to allow accumulation of ammon~um
Wo 93~8269 Pcr/uS92/09024
212~138
ions urithin tbe bound cells, followed by the incubation of bou~d ceL~ co~taining
accumulated ammoDium ioDs in the presence of ammonia and c;~bon dio~ude
such th~t selective hemolysis of ma~ernal blood cells occurs. These hvo
enrichment methods may be perfolmed sequentially, and in either order.
Within another aspect of the prese~t invention, a method is
pr~vided for enriching fetal ~ucleated, er~oid cells from matemal blood,
compAsing the steps of: incubating a sample of ma~ernal blood in the presence ofelythropoietin such that the fetal cells are enriched; incubating the enriched cells
with an immobilized ligand capable of specifically b;nd~ng to fetal nucleated,
10 elythroid cells under co~ditions and for a time sufficient to allow speci~Sc binding
of the ligand to the cells; and removing unbound blood products. Alternatively,
within another embodiment of the present invention, the enriched cells may be
immobilized by ~ncubating them with a first member chem~cally linked to a ligandcapable of specifically binding to the cells under conditions and for a time
15 sufficient to allow specific binding of the ligand to the cells, and adsorbing the
cells to an immobilized second membe-, the second member being capable of
billding to the first member with an affinily cor~ t of greater than about
108 M-1 . Wi~n yet ~ ~ther embodiment of the present invention, the enriched
cells may be imlmobilized by incubating the enri~ed cells with ~ first ligand
20 capable of speciScally bindLng to the cells under coIlditions aIld for a timesufflcient ts~ allow specific binding of the first ligand to the cells; incllbating the
sample with a first member che~cally linked to a second ligand capable of
specifically binding to the iirst ligand under conditions and ~or a time sufficient to
allow tbe second ~gand to bind to the first ligand; and adsorbing the cells to an
25 immobilized second member, the second member being capable of binding to the
fi~st member with ~ ~t~ cons~t of greater than about 10B M-1, Suitable
combiIIati~ns of ~rs~ ~nd second ligand are discussed in detail below.
In addition, ~nthin the aspects discussed above, the methods may
further comprise ~subsequent to removing the unbound blood products~ the steps
30 of: incuba~ng the bo-~d cells with ammoI~ia and ~hloride ions and a carbol~icanhydrase inhibitor under conditions and for a time suflïcient to allow
accumulation of ammonium ions within the bound cells; and incubating the cells
containing accumulated ammonium ions in the presence of ammoni~ and c~rbon
dioxide, such that selective hemolysis of maternal blood cells occurs.
Within otker aspects of the present inventio~ as an alternative to
first incubating a sample of maternal blood in the presence of eIythropoietin, the
cells are incubated with ammonia and chloride ions and a carbsnic anhydrase
wo 93/08-~69 . Pcr~us92/os~2
iIlhibitor uIIder condi~o~ d for a time su~cicnt to allow accumulalio~ of
ammo~um io~s wit~ thc bound c~lls, followet by i~cuba~os of the cells
containing accumulatcd ammor~ium ioDs i~ thc prescncc of ammo~a a~d carbo~
dio~ade, such that scle~ve hemolysis of matcr~al blood cells occurs.
S Within the prescnt i~ventia~, a ~ranc~ of ligands may be ut~d,
includi~g antl~dics, crythropoie~, a~d ~sfc~ Thc ligand may be
immobilized o~ any of a ~rane~r of sa}id supports, such as hollow fibe~s, bcads,magnetic b~ads, platcs, dishes, ~asks, meshcs, scrceDs, solid fibess~ membranes,a~d dipstic3cs.
Withi~ another aspect of thc inYe~on a method for c~nching fc~
proge~i~or cells from maternal blood is pronded compnsing the steps of
:~ ~: (a) incubating a samplc of mater~al blood ~th an ~obilized ligand capable of
speci~cally binding to fe~al progenitor cells ulldcr condi~ons, and for a ~me
sufficierlt to allow specific bindi~g of ~e ligand to t~e cclls, a~d (b) removing
15 uIlbou~d blood products such that thc fe~al progc~itor cells are cnrichcd Within
OIlC cmbodimenL prior to the stcp of incuba~ng, red blood cclls are removed ~rommatcrnal blood, for example by n~n~ing the matc~al blood over a ~icoll gradien~
Wi~ anothcr embodimcnt, subsequc~ to the StCp of removing thc unbound
blood products, the bound cells are i~albatcd in the presence of erythropoie~
Withi~ anothcr aspect of the prcse~t ~n~en~ion, a method is
provided for em;ching fetal progemtor cells ~om matcsnal blood comprising the
steps of (a) inalba~g a~ sample of matcrQal blood with a labcled ligand capablc
of specifically billdi~g to fetal progeDitor c~lls, under co~di~ons, arld for a ~e
sufficient to:allow:speci~ic biIl&g of thc ligand ts the cclls, (b)d~t~c~ing thepr~ence of the~ ligand bou~d~cells~ and (c) ~cpara~ng the liYand bouIld cells ~om
the unbou~d c~s,:~such that said fesal proge~itor cells iare cnnchcd. Wi~hini one
embadiment, p~ior to the stcp of i~ ba~g, red blood cells aFe removed from
mate~al bioo;~ ~y, for cxample, n2n~g tbe matcmal blood ovcr a Ficoll
~ent. Wi~ a~other cmbodime~e, 5UbSalUCalt tO the step of removing the
u~bound blood products, the bou~d cells asc iIlcubaud in the prcsence of
e~hropoie~ Withi~ the embodi~ients, the labcl is selec~ed from the group
ng of ~oresciein-isQthioqanate, phycoe~hrin, rhodamine iso~iocyana~e,
: ~ or other such highly ~uoresccnt moleQLes. I~ other e~nbodimcnts of the
~ invention, ~iC ligand ~ ~ibodv such as 1~8.
:~: 35 Withi~ another aspe~t of ~he ~reDi~on. a mcunod is provided for
en~iching fe~ progen2tor cells from mate~l blood com~rising, ~hc stcps ~f
~a) incubating a s~nple of materr~ blood with an immobi}iz~d ligand capable ot
Wo 93/08269 Pcr/uss2/o9o24
2122138 `~`~
specifically binding to cells other than fetal progeDitor cells, under condiffoIls and
for a time sufflcient to allow specific binding of the ligand to said other cells, and
~b) remc~ving the nonbound fetal progenitor cells, such that said fetal progenitor
cells are enriched. Within one embodiment, prior to the step of incuba~dng, red
S blood cells are removed from maternal blood by, for example, running the
maternal blood over a Ficoll gradient. Within a preferred embodiment, the ligandis a~ antibody.
Within another aspect of the present invention a method is provided
for typing chromosomes of fetal nucleated erythroid cells, comprising the steps of
10 (a) incubating the fetal nucleated erythroid cells in a media contaiI~ing
e~thropoietin under conditions and for a time sufficient to induce metaphase in
the cells, (b) f;xing the DNA of the cells~ (c) staining the fixed DNA such thatchromosomes may be obsenred, and (d) examining the stained DNA thereby
allowing the ~ping of the chromosomes.
These and other aspects of the present invention will become
evident upon reference to the following detailed description.
Detailed Desc~i~tion of the Invention
As noted above, within one aspect of the present invention methods
20 are provided for the enric~ment of fetal nucleated, erythroid cells from maternal
blood. Maternal blood colltains, among many other ~pes of cells, both adult and
fetal nucleated, e~ythroid cells. Througb the efforts of the present invention, fetal
nucleated, erythro~id cells~ may be enriched from as few as 1 in 106 in.maternalblood, to an enr~ched concentration of about 1 in 103, and preferably, to about 1
25 in 102. Wi~n the context of the present invention, nucleated el~rthroid cellscontain a nucleus and generally include e~ythroblasts as well as other erythroidprècursor cells.
Maternal blood may be obtained from a pregna~t female using
conventional techniques well Lnown in the art. Preferably, peripheral blood is
30 drawII from an e~ily obtainable source such as the antecubital vein ~the arm vein)
with conventional venipuncture techniques. Once the maternal blood has been
drawn, it may be frozen using conventional techniques, or stored at 4C for a
maximum of 4 to 7 days. Various anticoagulants may be added to the blood as
ne~essaly, including, among others, ACD, CPDA, EDTA, and Heparin.
The maternal blood is then subjec~ed to a selection method in
accordance wi~h ~he present invention, wherein preferentially enriched fetal cells
may be obtained urithout the need for further purif;cation, for example, by a
WO 93/08269 2 1 2 2 1 3 8 Pcr/vss2/o9o24
Fluorescence-Activated Cell Sorter (FA(~S). In general, ~he methods of tbe
presellt invention comprise the steps of: (1) incubating the maternal blood witheither an immobilized ligand, or a ligand which will subsequently be immo~ilized,
such that the ligand binds to and hence immobilizes the fetal ~ucleated, erythroid
S cells, (2) remo~nng unbound blood products, aIId ~3) preferentially enriching the
bound cells for fetal nucleated, erythroid cells. As noted above, these basic steps
may be performed in an alternative order, for example, comprising the steps of:
(1)preferentially enriching maternal blood for fetal nucleated, eIythroid cellsj~2) incubating the enriched cells with either an i~nmobilized ligand, or a ligand
10 which will subsequently be immobilized, such that the ligand binds to and hence
immobilizes the fetal nucleated, erythroid cells, and (3) removing unbound bloodproducts. The present invention may be perforrned utilizing devices as describedin an application entitled "I~unoselection Device and Method," U.S. Serial No.
07/513,S43, and an application entitled "An Apparatus and Method for Separating
15 Particles Using a Pliable Vessel," U.S. Serial Mo. 07/599,796, both of which are
incorporated herein by reference.
In addition, one may utilize an immunoaffinity column as described
in pending U.S. Application ~Attorneys Docket No. 200072.407) Serial No.
entitled "Improved ~pparatus and Method for Cell Separation"
20 (hereby incorporated by reference in its entire~). Briefly, within one aspect of
this application, a "cell separator" is provided, including a colurnn assembly for
separating target cells ~rom a sample fluid, the column assembly including a
colu~, a sample fluid supply bag and a fluid collection bag wherein the column is
provided for receiving the sample fluid ~om the sample fluid supply bag and for
25 separatillg ~e target cells ~om the sample ~uid and retaining the ta~get cells, and
wherein:the ffuid collection bag is provided for receivi~g the ta~get cells after
being released ~om the column, said cell separator comprising aIl agitation mea~s
for a~tating the contents of the column to assist in releasing the sample cells
retained in the column, the agitation mealls being responsive to a drive signal for
30 va~ying amount of agitation of the contents of the column to va~y the ra~e at which
he sample cells are released, column sensor means for providing a column signal
indicative of the optical densi~ly of ~uid flowing out of the column and into the
fluid collection bag, a column valve means response to a column valve control
signal for selectively enabling the fluid coming out of the column to flow into the
35 ~luid collection bag, axld a data processor means for controlling the operation of
the cell separator, the data processor means being response to the column signalfor providing the drive signal and the column valve con~rol signal to prevent
WO 93/0826~ P~r/uss2/~9o24
2122138 8
inadequ~te concentra~ions of the target cells from being collected. One
embod~ent of ~is invention is tbe OEPRAT~ I~n' cell sep~ratio~ system which
is available from CellPro0 (Bothell, Wash.).
Within one aspect of the present inve~tion, the mate~al blood is
S incubated with an immobilized lîgand capable of spe~ifically binding to ~etal
nucleated, erythroid cells ullder conditions, and f~r a time sufficient to allowbinding of the cells to the ligand. Generally, incubation of about 15 to 30 ~utes
at 4C to 37C is preferred. If the incubation step occurs as the cells are passed.
~ver a column, ~he flow rate should be suf~ciently slow to allow the cells to bind.
10 Pre~rably, the cells should be allowed at }east 15 ~utes in which to traverse the
bed of the column.
As noted above, the ligand should be chosen such that it is capable
of specifically bLnding fetal mlcleated, elythroid cells. Within the context of the
present i~vention, the ligalld is def;ned to be "specifically binding" if it is capable
of binding to fetal nucleated, erythroid cells, but not to more than about 10~o of
the matemal blood cells. The relative percentage of bound fet~l nucleated,
elrythroid cells to other cells m~y be readily de~e~ined by analysis w,ith a specific
marker such as Alpha Fetal Protein (AFP). For example, at a~y point within ~e
methods discussed below, the relative percentage of fetal cells to rnaternal cells
:: 20 may be de~ermined with either glucose o~dase or fluorescein conjugated anti-
AFP an~body. See ~ ~ulozilc and I. H. Pawlowitzki, "Fet~l Cells in the Maternal
Circulation: Detection by Direct AF~-Immunofluorescence," ~Iuman Genet.
62:221 (1982). This determination is pr~erably performed af~er ~vo Dl more
pu~ification steps.
2~ IigaJlds whic~ spe~ifically bind to fetal nucleated, erythroid cells are
knouql in the ar~"ncluding e~hropoietill (Amgen, ~ousand Oalcs, Calif.),
~: ~ transfe~i~ (Sigma (:hemieal Co., St. Louis, Mo.) and sele~ed a~tibodies.
~onoclonal antibodies whicb spe~ically recognize Ilucleated erythroid cells are
particularly preferred. Monoclonal antibodies to nucleated e~ythroid cells such as
30 allti~ ferrin receptor antibodies may be ob~ained from conYentional suppliers(~ecton Dickinson Immunocytometry Systems~ Mowltain View, Calif.~.
Alternatively, monoclonal anti-erythroid antibodies, such as EP-1, may be
produced using techniqlles well kn~ the art. See Yokochi ~t a~, "MoIloclonal
Antibodies Detecting Antigenic Determinants With Res~ricted l~xpression On
35 Erythroid Cells: From ~he Erythroid Committed Progenitor Level to the Mature
E~ythroblast," Blood 63:1376 ~1984); see also Heddy Zola (ed.), ~Ionoclonal
Ar~ibodies: A Manual of Techni~ues, CRC Press, Boca R~on, Fla. ~1987).
WO 93/~8269 Pcr/uss2/o9~24
2122138
Briefly7 cells may be generated for ~nization from fetal liver clonal erythroid
cultures, and e~iched for progen~tor ~lls. Thu~, ~he poplllati~n o cell~ used for
aIltigens aIld for plimary screening may contain ~mmature eIythroblasts,
erythrobla~ts of all intermediate degree o matuA~y, and presumably, progenitor
cells of BFIJ-E arld CFU-E ~pes. These cells may be used for intravenous
immuniza~lio~, followed by removal of the splee~ and fu~ion of the spleen cells
with a myeloma line such as NSI, using standard techIIiques. The result~g fused
cells, or hybridomas, may then be screened ag~t the above-described cells using
conventional techniques. See Yokochi et a~, supra.
The e~tire specifically binding antibody need not be used as the
ligand. More specifically, only the binding region of the an~ibody is necessa~y to
specifically bLnd fetal nucleated, eIythroid cells. Thus, antibody ~agments such as
Fab or F(ab'~2 ~agments may be used within the presen~ inveIltion. Additionally,the binding regions of the specifically binding antibody may be incorporated into a
15 new protein, which may be used as the ligalld. See Reichmann et ~, "Reshaping:~ Human Antibodies For Therapy," Na~ure 332:323-327 ~1988); Verhoeyen et al.,
"Reshaping Human Antibodies: Grafting an Antilyso~yme Ac~ y," Science
239:1534-1536 (1989); and Robe~s e~ a~, "Generation of an Antibody ~th
EnhaIlced ~i~y and Specifici~ ~or its Antigen by Pro~ein l~nginee~g," N~ur~
: 20 328:731-734 (1987).
Within ~he present invention, the ligand is immobilized in order to
allow separation: of bound cells from other blood products. Many suitable
supports are well l~own in the art alld include, amo~ others, hollow fibers
(~con (:orporatiorl, Danvers, Mass.), beads (Polysciences9 Warrington, Penn.),
25 magnetic beads (Robbin Scientific, Mou~tain View, Calif.), plates, dishes andflasks (Corn~g Glass Works, Corning~ N.Y.), meshes (Becton Diclcinson,
Mountain View, ~.), screens and solid fibers (see Edelmall et al., U.S. Patent
No. 3,843,324; see also Kuroda et al, U.S. Patent No. 4,416,777), membranes
~Mi}lipore Corp., Bed~ord, Mass.~ and dipsticks. A variety of different sources
30 exist for supports other than ~ose designated. Particularly preferred is a support
such as Biogel P-60n' (BIC)RAl:), Richmond, C:alif.). Biogel P-60n' is a porous
polyacrylamide hydrogel bead. The beads are generally spher~cal, on average
about 250 microns in size, and have an average pore size which excludes
molecules larger than approximately 60,000 daltons.
3~ A va~ie~ of methods may be used to immobilize the ligand onto a
support. For example, a ligand, such as an antibody, may be direct~y coupled to
the support by vari~us methods w~ll known in the a~t. 3ee J. K ~marL, Methods In
W O 93/08269 PC~r/US92/09024 2 1 2 2 1 3 8
Enymology, Vol. 34,A~qnity Tech~es, EnymePurificatio~ PartB, W. B. Jakoby
and M. Wilchek (eds.), Academic Prexs, New York, p. 30 (1974); see also
M. Wilchek and W. Bayer, nThe Avidin-Biotin Complex in Bioanalytical
Applications,"Ana~t. Biochen~ 171:1-32 (1988). These methods include the use
5 of glutaraldehyde, carbodiimide, carbonyl diimidazole, cyanogen bromide, tosyl~hlo~ide, biotin/avidin, and biotin/streptavidin. Once the ligand has been
immobilized onto the support, the maternal blood may be incubated with the
immobilized ligazld under conditions, and for a time sufficient to allow binding of
the ligand to the cells. Within the context of the present invention, suitable
10 conditions for binding to occur include incubation in a physiological buffer at
about 4C to about 37C. Particularly preferred temperatures range ~om about
4C to room temperature. The time of incubation depends on the afflnity and
avidi~y of the ligand for the cell, and may be readily determined. Generally,
incubation for about 15 minutes to one hour is preferred. Following incubation,
15 unbound blood products may be removed, and fetal cells enriched using the
~; ~ methods describe~ herein.
Within another aspect of the present invention, a sa~nple of
maternal blood is incubated under suitable conditions with a ligand which is
chemically linked to a first member, and then adsorbed to a second member
20 which is immobilized on a solid support. The first member should be capable of
binding to the second member with an aflini~y of greater than about 108 M~1
Many suitable f;rst member-second member binding pairs are well l~own in the
art. These include, among others, biotin-avidin, biotin-streptavidin, bio~ytin-
andin, biog~tin-streptavidin, methotrexate-dihydrofolate reductase,
25 S-fluorouracil-thimydylate synthetase, riboilavin-riboflavin binding prote;n,antibody-protein A, and antibody-protein G. In a preferred embodiment, the firstmember is biotin and the second member is avidin.
Either member of the above described binding pairs may function as
the second member, with the complementa~y member functioning as the first
30 member. Furthermore, combinations of the first member-second member binding
pair may be employed. For example, biotin may be linked to the ligand, as well as
adsorbed to the suppon. The cell, ligand, biotin-eomplex and biotin, support-
complex may then be bound together through an incubation step with andin.
Avidin is multivalent, permitting the ~ormation of a cell, ligand, biotin, avidin,
35 biotin, support-complex which immobili~es the cell.
Within one example of this embodiment, a sample o~ maternal
blood is incubated with a biotinylated antibody under conditions and for a time
WO 93tn8~69 21 2 2 ~ ~ 8 Pcr/us92/oso24
11
sufficient to allow binding to occur. 'rbe sample is then incubated wi~h, or passed
over, a support which contains immobilized andin. Cells which are coupled to thebiotinylated antibody are adsorbed to the immobilized avidin, thus allowing
separation of cells from unb~und blood products. Subsequently, unbound blood
S products may be removed, a~ld fetal cells enriched using methods described below.
Within yet a~other aspect of the present invention, a tw~step
method is used to immobilize tbe fetal nucleated, erythroid cells. Brie~y, ~ first
ligand is incubated with a sample of the maternal blood under suitable conditions
as described above. Subsequently, a second ligand which has been chemically
10 linlced to a first member is added. 'rhe second ligand is capable of bind~ng to the
first ligand. The cell, first ligand, second ligaIld, first member-complex may then
be adsorbed onto an immobilized second member, t~us allowing the separation of
cells ~om unbound blood products. Representative examples of the first member-
second member binding pair have been discussed above. Representative
15 ~ examples of the first ligand include eIythropoietin, transferrin and selected
antibo~es. Once the first !igand has been selected, the second ligand is chosen
such that it specifically recogluzes and binds to the hrst ligand. Within a preferred
embodimenL the second ligand is an antibody, for example, an anti-e~thropoietin
(Teny Fox Laboratory, Vancouver, B.C., Canada), anti-transferrin (Chemicon
20 Intl., Inc., Teme~ula, C~alif.), or anti-immunoglobulin antibody. Anti-
immunoglobulin antibodies may be prepared using techniques well hlown in the
art, or may be ~ined ~from conventiona~ sources, including, among others,
Sigma Chemical Co., St. Louis, Mo., and Becton Dickinson Immunocytometry
Systems, M~untain ~riew, Calif.
25 ~ ~ Within a preferred embodiment, the first ligand is an antibody
which specifically~recognizes fetal Ilucleated, erythroid cells such as an anti-; transferrin receptor antibody (Becton Dicldnson Immuno~ytometry Systems,
Mo~tain View, C~lif.). The antibody is ~ncubated with a sample of matemal
~, blood. A biotinylated anti-immunoglo~ulin alltibody, such as biotirlylated goat
30 anti-mouse lgG ~the second lig~d which is cbemically linked to a first member) is
then added and iIlcubated with the sample. The sample is then incubated u ith, or
passed over a bed of material which contains the immobilized second member, in
this case, immobilized avidin. The cell, antibody, anti-immiunoglobulin antibody,
and biotin-eomplex will be adsorbed to the immobilized avidin, thus allowing the35 subsequent removal of unbound blood products.
As noted aboYe, once the cells have been immobiliz~d, unbound
blood products may be removed. In one embodiment, the immobilized cells are
WO 93/08269 Pcr/us92/09024
j~ ~,,
2122138 12
rinsed with a physiological bu~er9 thereby removing the unbound blood products~
Va~ious methods may be used to ~inse the immobilized cells, depending upon the
type of support chosen. These methods include, among others: washing or
flushing the support; magnetically attracting the support out of solution, followed
S by resuspension in a physiological buf~er; and centrifuga~ion followed by
resuspension. Various physiological buffers are also well known in the art,
including PBS, PBS plus albumin, such as Bovine Serum Albumin ~BSA), normal
saline and cell culture rnedi~.
Once unbound blood products bave been removed, bound cells may
10 be preferentially enriched ~r fetal nucleated, e~ythroid cel~s. As noted above, at
least hvo alternative methods may be used either separately, or together. Lf thetwo methods are performed together, either method may be performed first.
Within one embodiment the bound cells are cultured under selected culture
conditions in the presence of elythropoietin (Amgen, Thousand Oaks, Calif.). SeeEmerson et a~, "Developmental Regulation of Erythropoiesis by Hem~topoietic
Growth Factors: Analysis on Populations of BFU~E From Bone Marrow,
Peripheral Blood and~ Fetal Liver," Blood 74(1):49-55 ~1989); see also Iinch et a~,
~;: "Studies of Circulating Hemopoietic Progenitor Cells in Huunan Fetal Blood,"
Blood 59(5~:976-979 (1982). Selected culture conditions generally include growth20 in standard cell c~lture ~media, without any other ~ytoldnes other tha
:e~hropoietin. This preferentially allows fetal, but not maternal, nucleated
erythroid: cells to grow. Pa~ticularly preferred media includes Iscoves' Modified
Dulbecco's Medium (Gibco, GraIId Island, N.Y.) containing a final concentration
of 20% fetaI bov~ne serum, and 2 U/ml puri~ed unnaly human EPO.
Within the second method, fetal nucleated, exy~roid cells are
preferentially nriched based upon tb,eir uptake of ammo~ium ions and the
~;: selective hemolysis of maternal blood cells, or more speciiïcally, of maternal
elythroid cells. See genenalb Jacobs and Stewart, '~he Role of Carbonic
:~ ., Anhydrase in Certain Io~ic Exchallges Involving the EIythro~yte,".~. Ger~ Physio~
25:539-552 (1942); and: Maren and Wiley, 'Kinetics of Carbonic Anhydrase in
Whole Red Cells as Measured by Transfer of Carbon I:)io~de and Ammonia,"
Molecular Pharmacolo~ 6:430440 (1970). Briefly, the cells are incubated in the
~: : presence of ammonia alld chloride ions and a carbonic anhydrase inhibitor under
conditions and for a time sufficient to allow accumulation of ammonium ions
35 within the cells. 'rhis generally takex approximately 5 to 30 minutes. The cells are
then incubated in the presence of ammonia and ca~bon dioxide such that selectivehemolysis of maternal blood cells occurs.
WO 93/08269 2 1 2 2 1 3 8 PCI/U~92/09~24
13
Withi~ the context of the present invention, many compounds may
provide sui~able sources of ammonia, shloride io~s and carbon dio~de. For
example, sui~ble sources of ammonia include, among others, ammon-a and the
ammonium salts. Suitable sources of chloride ions include, among others~ NaCl,
S KCl, MgCi2 and CaC12. ~uitable sources ~or carbon dio~nde anclude9 among
others, carbon dioxide iII solution, carbonate and bicarbol~ate.
In addition, many carbonic a~hydrase inhibitors are known in the
art3 including, among others, most monovalent ~ons such as ~yanide and cyanate,
monovalent sulfides, sulfonamides, and acetazolamide. See Lindskog e~ a~, The
10 Er~ymes 5:587 (1971); Ward and C~ull, Arch Biochem. Biophys. 150:436 (1972~;
and Pocker alld Watamori, Biochem. 12:2475 (1973). The carbo~ic anhydrase
inhibitors should b~ selected so as to functi~n under physiological conditions.
Preferred carbonic anhydrase inhibitors include sulfanilarI~ide and acetazola~ide.
Bound cells may be released by various methods either subsequent
15 to, or prior to en~ichment. If bound cells are released prior to erllichmen~, they
may be subsequentl~ enriched using the above-described methods. Various
~: methods are known L~ the art ~or releasing cells. Within one such method3 cells
may be cul~ured with or without 6~ytoldnes. C~tokines, such as II~2, can cause
prol~eration of cells, or cha~ges iII the su~ace characteristics of ~e cells, such
;: 20 that the cells or their progeny are released i~om a support. Within another
me~hod, cleavage of the li~and or of the cell-ligand bond may release the cell.
Various cleavalble lig~ds and cleaving enzymes are Icnown in the ar~9 including
among others, papain alld t~ypsin. Within yet aIlo~er method, the cells may be
: : released by mes~aI~ical, ~avitational or electromagnetic forces. A particularly
25 preferred method i~ mechanical agitation, for ~xample, by agitation of the beads
thr~ugh pipett~g, stimng9 sh~g, vibration, or sonic~tion.
~ i ~ithin aIlo~her aspect of the present invention, a method is
: provided wherein the cells are i~rst en iched, ~ollowed by adso~ption of fetal
;; ~ I ~ucleated, erythroid cells and removal of unbound blood products. Briefly, fetal
30 cells m~y be enriched by hrst incubating materllal blood wi~h a~noI~a and
chloride ions, and a carbonic anhydrase inhibitor. After the cells have been
incubated under conditions and for a time sufficient to allow accumulation of
anLmonium ions, the cells are ~eated with ammonia and carbon di~xide such that
~: selective hemolysis of maternal blood cells occurs. Fetal cells may also be
: 35 enriched by incubation u ith e~hropoietin as discussed above, or by a combined
treatment using both erythropoietin and the method discussed above wherein $he
Wo 93/08269 PCr/US9~/09024
212~ 138 ";
14
cells are treated with ammonia and carbon dio~de. Furthermore, as discussed
above, the ~NO methods may be performed in either order.
The enriched cells remaining after the method(s) described ab~ve
may then be ~obilized usi~g any of the above discussed methods, including:
S (1) an ~obilized ligall~ w~ich specifically binds ~o fetal nucleated, e~roid
cells rem~ining after enrichme~t, (2) incubatiIlg cells rem~g after enrichment
with a hrst member linked to a ligand, the ligand being capable of specifically
binding to fetal nucleated, e~ythroid cells, followed by adso~p~Lion of the cells to an.
immobilized second member, the second member being capable of binding to the
10 fîrst member with an ~ity constant of greater than about 108M-1, and
(3) incubating c~lls rem~ng after enrichment with a first ligalld, followed by
incubation with a second ligaIld which is chemically linked to a first member which
is capable of binding to the first ligand under conditions and for a time sufficient
to allow the second ligalld ~o bind to the first ligalld, followed by adsorption of the
cells to an ~obilized secolld me~ber, the second member being capable of
binding to the first member with an affinity eo:nstant of greater than about
o~M-l
~: Subsequent ~o ~obilization of the cells~ unbou~d blood products
m~y be removed using methods described above. If the cells were initially
en~iched using elythropoietin, subsequent to removing unbound blood products,
the cells may be treated using the method wherein ammonia and carbon dioxide
are used. Similarly, if these cells were initially treated with the method wherein
~onia alld c~bon dio~ide are used, s~sequent to remov~ng unbound blood
products, the cells may be treated with eIy~ropoietin.
Fetal cells which haYe been enricbed from maternal blood may be
chara~terized by incubating the preferentially enriched ~ells with a marker
c~pable of selectively binding to ~etal cells. A marker selectiYely binds to ~etal
cells when its presence on the target cells is greater ~han 1~fold more than thequantity on maternal cells. Various markers are knowD in the art, including, for30 example, ~tibodies to Alpha Fetal Protein ("AFP"). See Kulozik et al., "FetalCells in the Maternal Circulation: l~etection by Direct AFP-
Immunofluor~ssence," Human Genet. 62:221-224 (1982), or antibodies to antigen
"i". See Y.W. Kan et al., "C: oncentration of Fetal Red Blood Cells From a Mvsture
of MaterIlal and Fetal Blood by Anti-i Serurn," Blood 43(3):411~15 (1974).
35 Antibodies to the fetal cell marker may be labeled using techniques well known in
the a~t and used to detect the presence of fetal cells.
WO 93J08269 2 1 2 2 1 3 8 Pcr/uss2/09024
~5
Within a prefelred embodimellt of the present ~vention, liga~d is
removed from the cell surface through ~he ~ethod described in a related
application (U.S. Serial No. 07/513,056) elltitled "Methods for Removing Iigallds
from a Pa~ticle Surface", which is Lncorporated hereiIl by reference. Suc~ removal
S may be particula~ly advalltage~us prior to characte~tion, as discussed above.
Within a~other aspect of the invention, a method for er~iching fetal
progenitor cells ~om mate~nal blood is provided comprising the steps of
(a) incubadng a sanlple of mater~al blood wi~h axl immobilized ligand capable obinding ~o fetal progenitor cells under conditions, and for a time suf~cient to allow
10 specific binding of the ligand to the cells, and (b)removing u~bound blood
products such that the fetal progenito~ cells are enriched. Within the context of
the present iIlvention, fetal progenitor cells includes totipotent hematopoieticstem cells as well as early progenitor cells such as colony forming cells (CFCs).
Representative examples of CFCS include CFIJ-E, CFIJ-G, CFU-M, CFU-GM,
15 CFIJ-GEMM and BF~J-E cells. Given the ~act that generally as many as one-
third to two-thirds of CD 34 positive cells may be stem cells or colony-~orming
cells, it should be understood that when CD 34 ceL~ are ~oncentra~ed or purif;ed,
fetal progenitor cells a~e lik~wise unders~ood to be concentrated or purified~
As noted aboYe, the sample of maternal blood is Lncubated with an
20 immobilized ligand c2lpable of binding to fetal progeDitor ce}ls. Wi~hin the
context of the present invention, a ligand '~inds to" fetal progenitor cells if it
:: recognizes a~ tigen associated with either both adul~ d ~etal cells or only fetal
;~ ~ progenitor cells. A representative antigen in ~lhis regard is the (:D-34 antigen.
Representative examples of antibodies which spe~;~cally recognize tbe CD 34
25 aIltigen include~ 10 aIld lHP~2, (BeCt~n-D1C1~nSOn~ MONntain View, Calif.~,
QBEND-10 (Quantum Bi~systems, ~ambridge, U.K) and 12~8 ~Cellpro~, Bothell,
W~h.). ~ ligaIld may be utilized in the above~described devices and methods
in order to in~obilize tbe i~etal progeDitor cells. After incubation, the unbound
blood products are removed as described above, ~uch that the ~etal progenitor
30 cells are enriched. Within the context of the present invention9 cells are
: ~ "enn~hed" if gre~ter than 0.001% of the cells a~e fetal progeI~itor cells. Preferably,
~:~ the fetal progenitor cells are ennched to greater than .1% and particularly, to
~: greater than 1~ of the cells present.
Within another aspect of the preserlt invention, a method is
35 provided for en~ching fetal progen:itor celis from maternal blood comprising the
steps of (a) incuba~ing a sample of mate~al blood with a labeled lig~nd capable
of specifically biIlding to ~etal progerlitor cells under conditions and for a time
.. . . .
.... . ..
.. .
W~3 93/~)X2~9 PCr/U!;92/a~024
` 16
slaffi~ent to allow specific binding of the liga~d to the cells, (b) dete~g the
prcse~cc of the ligant bou~d cells, and ~c) scpara~ tbe ligand bou~d cells from
the unbo~d cells, such that thc ~tal proge~tor cells are e~r~ched. Wi~hi~ this
aspect of the ~nYcntioD, ceL~s are ~Ilaubated ~nth a liga~ld sucb as that descrl~ed
S abovc w~ich is c~able of spccifically bindi~g to fe~l progcnitor cc~ls, except that
rathcr thaIl bcing ~obilized, this liga~d is labelcd. Yarious Iabcls may be
u~lized withi~ the co~sext of the pres~t ~c~io~, although ~uorescem-
isothocya~atc? phycoe~, rhodam~e isothios:yanate, or other such highly
fl~uoresce~t molccules are par~cularly prefe~Ted.
1~ l~ough usc of ~ow cytometry (FACS) labeled ceLs may then be
dete~ed, and separated from no~-labeled (or a n~n-ligand bou~d) cells, such that.etal progcnitor ceLIs are e~Tiched.
Within o~e embodimcnt of the i~re~ion, prior to thc step of
incubati~g, red blood cells may be removed from m~ter~al blood, for example, by
15 ~g the materIlal blood over a Ficoll ~radie~ Withi~ another embodim~t,
subse~ue~t to the step of rcmo~ be ~bou~d blood produc~ thc bound cells
arc incubated i~ the prescnce of e~hropoie~n u~li~g culture me~hods
des~bed ab~c.
Wi~n a~o~er aspec~ of tbe ~v~ on, a method is provaded for
20 e~nchi~g fetal p~ogeni~or ceL~ m mate~ blaod, compnsi~g the steps of
(a) i~cuba~ng a samplc of mat~n: al blood wi~ i~obilized ligand capable of
spec~ically bi~g to ceLs o~her than fetal proge~itor c~lls under condi~iorls andfor a ~me suf ;cieat to ~ow specific bi~ding of thc ligand to the othe~ cells, and
(b) remo~i~g the nonbound ~tal progen~or cells, such ~hat the fe~ progeD~tor
25 cells are e~ichcd. As ~oted abo~e, CD 34 is a cell-surface an~gen that may befou~d o~ f~tal proge~itor cells. Ma~y ce~face a~gc~s howc~vcr, can not be
found o~ proge~or ceLs, aIld thu~ maybc udlized to dcp}et~ a samplc of matcmal
bl~d ~rom ccLl othcr than fetal proge~tor c~lls 3Rcpresc~ ve examplcs of
such arl~igeDs ~cllldc Ia, Glycopho~ CD 3, CD 19, CD 11, CD 1~, CD 33, and
30 CD 45. Iig~ds, such as a~ibodies, whieh specifically bind tO such an~gens maybe purchased ~orrl commercial supplie~s such as Becto~-Dic3c~soD, Mountain
Vi~w, Calif.
Within one cmbodime~t of this mven~o~ prior to the s~ep of
incuba~g, red blood c lls arc removed ~um mater~al blood ~, for example,
35 n~i~g the maternal blaod over a Ficoll gradient.
1~:
W(~ 93/08269 2 1 2 2 1 ~ 8 PCr/US92/U9024
USBO~FErAL CE~LLS
As described herein, enriched fetal cells have a Yarie~ of uses. For
example, through in si~u hybridization the presence of a selected genetic material
5 may be detected in a fetal cell. Similarly, DNA or RNA amplification m~y also be
used to detect a selected genetic sequence in fetal cells. The methods of the
present invention are partic~arly useful for fetal cells which are enriched to such
an ~tent that they are ~menable to chromosomal typing by conveIltional
cytogenetics techDiques wherein spreads of the cells' chromosomes a~e examined
10 under a rnicroscope.
In sih~ hyb~id~tion may be used as a method for detecting the
presence of a selected genetic material within cells. See Pin:kel et a~, Proc Natl.
Acad. Sci U5A 85:913842 (1988); see a~so Hopman et al, "Detection of Numerical
~hromosome Aberrations in 13ladder Cancer by In Sitll Hybridization," Am. J. of
15 Pa~h 135(6):1105-1117 ~1989~. Briefly, the genetic material associated with the
preferentially enriched cells is first exposed usi~g teehniques well known in the
art. The genetic material is then incubated with a labeled probe capable of
specifically hybridizing to ~he genetic material under eonditions alld for a time
SUf~CieI1t tO a11OW hY~ atiOn tO OCCUr. See, for eXamP1e, K E. DaVieS, ~uman
20 Genetic JDise~ses, IRL PreSS, V~(3~, U.K (1986). Fina11Y, the PreSenCe Of thehYb~idLZed 1abe1ed PrObe iS deteCted. Within a Preferred embOdiment~ the genetiCmateria1 iS den~tUred after the SteP Of eXPOSing. Wit11in the COnteXt Of the PreSent
inVen~on:~ genetiC materia1 inCludes WhOle Chr~mOSOmeS, DNA and :RN~
; ~ ~ V:ariOUS ~etbOdS are alSo We11 knOwn jn the art fOr amplifying and
25 deteCting genetiC materia1. FOr eXamPIe, if PreSen~, a Se1eCted genetiC SeqUenCe
maY be amP~ ed USiDg te~hniqUeS We11 knOwn in the art, alld then PrObed fOr
presence of that se~uence. See ~og~ et al, "1~ Improved Me~hod For Prenatal
Dia~osis of Genetic Diseases by ~alysis of ~nplified DNA Seguences," The
New Eng. J. of A~e~ 317~16):985-990 (1987); see also Witt and Erickson, i'A Rapid
30 Method for Detection o Y~Chromosomal DNA from Dried Blood Specimens by
~e Polymerase Chain Reaction," Human Genet. ~2:271-274 (1989). Methods for
amplification include Polymerase Chain Reaction ("PCR") (see Mullis et al, U.S.
Patent No. 4,683,195; Mllllis et ~, IJ.S. Patent No. 4,683,202; and Mullis et a~,
U.S. P~erlt No. 4,800,159, which are incoIporated herein by reference), and RNA-
3~ based ampli~cation techniques. See Iizardi et al, Bio/'rechnology 6:1197-1202(1988); Kramer et a~, Nature 339:401-402 ~1989); and Lomeli e~ ~, Clinical
Wo g3/~82~9 Pcr/uss2/o9o2~
212213~ ~
1~
Chemistry 35(9):182~1831 (l9g9); see also Kramer et al, U.S. Patent
No. 4,786,600, which is inco~porated herein by refere~ce.
PCR is the most commoDly used method for ampli~g DNA
sequences. Briefly, amplification entails adding the appropriate primer(s),
5 er~mes aIld ~ucleotides i~to a reaction mix~ure, ~ollowed by several (2~80)
s~ycles ~f denatura~ion and ~nealing i~ order to ampli~ the small amount of
ta~get DN~ The DNA mixture is then separated by electrophoresis and
hybridized with a labeled probe to detect the presence of the target sequence ofDN~
The preferentially enriched fetal ce}ls may also be chromosomally
typed. See ~Iuman Cytogenetics, D.E. Rooney and B.H. Czepulkousl~ (edsO), IRL
Press, (:)xford, U.K (1986). Briefly, within a preferred embodiment, a sample
containing at least 1 fetal cell in 105 other cells is cultured for 2-6 days in Iscoves'
Modified Dulbecco's Medium (IMDM) (Gibco, Grand Island, N.Y.) contair ing a
15 final co~ceIItration of 20% ~etal bo~7ine serum (FBS) (HYCLONEn', Log~ IJtah)and 2 U/ml of highly pu~i~ed recombinant erythropoie~in (Terry Fox Laboratoly,
Vancouver, B.C., ~da) i~ order to increase the proportion of cells in
metaphase. The cells are then incubated in colcemid~ fixed, attac~2ed to
mi~oscope sl;des, t~psin-treated and stained with Wright's stain. The slides may20 then be sca~ned microscopically for abnormal chromosomes.
~: Preferen~dally enriched fetal cells may also be utilized
therapeutically. For example, within one aspec~ of the invention, a gene may be
inserted into a retrovirus, and the re~ro~nrus utilized to infect fetal cells which are
de~ciellt in this gene. The infected fetal cell may then be administered to the
25 fetus ~or another indi~idual) in order to remedy the genetic de~ect. Diseaseswhich may be treated in this manner are mlmerous, including for example ADA~
sickle cell ~mia, Thalassemia, a~d S~. Methods for inserdng genes into
retro~riruses are also well documented (see, for 0~unple, WO 90/01870, WO
88/03167, WO 88/09670, WO 89/07150~ WO 89/11539 a~d WO 89/09271, all of
30 which are incorporated by refere~ce).
Fetal cells may also be utilized as universal donor cells. Briefly,
because fetal progenitor cells do not c~eate as strong an immune reaction in
recipients as do progenitor cells ~om adults, they can be transplanted with fewer
side ef~ects. These fetal progenitor cells can thus be used for transplant to correct
35 inherited metabolic diseases in recipients. By expanding their rlumbers in culture,
these fe~l progeI~itor cells may even be used in place of a bone marrow transplant
following myeloblative therapy for ~ancer.
w~ 93/08269 2 1 2 ~ ~ 3 ~ P~r/US92/09024
19
Preferent;ally7 enri~hed fetal cells may also be made oncogenic ~n
order ~o stud~r the pro~ess of such oncogel~ic cells, or ~n order to simplify
e~ansion and maintenance of fetal cells for rese~rch or diagnostic purposes.
Represen~ative traIIsorming ge~es that may be ~erted i~to fetal cells (for
S example, by retro~ses as disa~sed above) include SV40, Ti, myc, ras and src.
Preferentially, emiched ~etal cells may also be cryopreserved for
future researeh, or i~or ~uture therapeutic use. BAefly, methods for freezi~g stem
cells are descnbed in a pendi~g application en~tled "Method for ~eezing
e~lgrafting c~lls" (At~orney~s Docket No. 200072.409). These methods may also be10 utilized to ~eeze fetal progenitor cells.
The following examples are offered by way of illustration~ and not
by way of limitation.
EXAMPLES
E~LE 1
Carboxylation of a Polyac~ylamide Gel
Seventee~ grams of dIy Biogel P-60n' ~5~100 mesh (wet), coa~se
20 beads) (BIORAD, C~atalog No. 15~163Q Richmond, Cali~ are added to 1.5 1 of
0.5 M N~ICO3/0.5 M Na2CO3. The pH is adjusted ~o 103 with NaOH and
care~ully stirred with a mixer ~R:ZRl~ C~famo, Wiarton, Ontario, Canada) so asi
not to damage the beads for appro~mately 20 to 30 ~utes. The mixture is then
placed iIl a 6ûC water bath. After the mixture reaches a temperature of 60C, it is
25 incubated for an addi~onal 2 hours (at 60~C) with occiasional stirring. The mixture
is then remoYed from ~he water bath, and placed iI~ an ice bath to bring the
mixture temperature down to room temperature.
The beads are washed several times with distilled or deionized
water, followed by severial wash~gs of PBS using a coarse glass filter connected to
30 a vacuum. ~he ~rboxylated gel may be stored iIl PBS at 4C~ and is stable for up
~o one year if sterilized or sgored with a preservative.
WO 93/~8269 Pcr/U~92/~9024
2122 1~8 ~ `~
~XAMPLli 2
Avidin Conjugating the Carba~ylated Biogel
PBS is first removed ~om a measured amount of ~rboxylated
S Biogel by filtering with a coarse glass filter connected to a vacuum. ~lle gel is
then equilibrated iIl distilled or deionized water for 15-30 minutes. EquilibratioIl
in wat~r causes a~ expa~sioll of the gel to a volume of about 4 times its previously
measu~ed amount. The gel is resuspended in 10 ml of distilled or deio~ed water
for each ml of gel ~as originally measured i~ PBS~.
T~ mg of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC-HCl) ~Sigma Chernical Co., Catalog No. E7750, St. Louis, Mo.) is added ~or
each ~ o~ gel as originally measured. The pH is rapidly adjusted to 5.5 by
dropwise addition of HCl. Care is taken to maintain the pH at 5.5; pHs of less
tha~ 5.0 or greater than 6.0 result in significantly less a~vation of the Biogel. The
: 15 mixture is stirred fo~ five mi~utes.
Avidin (International En:ymes, Inc., Fallbrook, Calif.) is diss~lved
at: a conceIltration of be~veen 10 ~nd :100 mg~ml in deioDized water. Next, 1000g ~f avidin is rapidly added for each ml of gel (as origiIIally measured in PBS~.
The:~mixhlre is stirred for 1.5 h~rs. N~xt, 2 M glycine is added to give a final20 conce tration ~0.2 M glycine in the~ nure and stirred for an additional 1 hour.
~ be gel is washed with several volllmes of P~3S using a coarse glass
er~ a~d vacuum, and stored in PBS at 4~. The gel is s~able for ~pproximately
one year.
, ~
EXAMPLE ~
Immunoadsorption of Maternal Cells With The Two-Step Method
, ~
P~PARATION OP ( ~ELLS
Twen~ milliliters of blood is obtained rom a pregnant female and
30 su~e~ded in ~ equal volume of PBS with l~o Bo~ine Serur~ umin (BSA)
(Sigma Chemical CO.? St. Louis, Mo.~ in four 50 ml centrifuge tubes. ~he number
:: : of nucleated cells/ml i~ counted in the A~ollowing manner A~ 50 ~1 aliquQ~ of
mixed3 articoagulated whole blood is diluted into 3 mA of a 3% acetic acid
SolutioAA. A~fter VOA texAllg~ 7 ,ul samples OA dAluted blood are loaded irto each of
: 35 t~vo chambers of a: hema~ytometer. After about 3 minutes to aAlow settling of the
cells, the nuclei of cells, which are almost all lysed by the acetic acid, a~e counted
in the f~ur ruled A~Aelds per chamb~r of a hema~ometer with improved Neubauer
WO 93/0826~ 2 1 2 .~ 1 3 8 Pcr/vss2/o9o24
21
rulings (VWR Scientific, Sa~ Frallcisco, Calif.), each field represendng 0.1 x 10-3
~1 sample volume. The average number of nuclei per field is multiplied by a
dilution factor of 61 x 104 to calculate the number of nucleated cells per rnilliliter
of whole blood. If the average is less than 10 nuclei per ~eld, the procedure is5 repeated, except 50 ~l blood is diluted into only 1 ml acetic acid, with a resulting
new dilution factor of 21 x 104.
Each tube of diluted blood is underlayered with 5 ml of Histopaque
1077 (Sigma Chernical Co.) and centrifuged at 700 x g for 15 minutes at room
temperature. Cells at the interface are collected and washed once in PBS plus 1
10 BS~ l~e pellet is resuspended in 100 ~l in PBS plus 1% BSA.
Twenty micrograrns/milliliters of anti-trallsferrin receptor antibody
(Becton Dickinson, Imrnunocytomet~ Systerns, Mountain View, Cali) is added
to the rnixture and incubated for 15 minutes on ice. The cells are then washed
once with 4 ml of PBS plus 1% BSA and centrifuged at approximately 400 x g for 515 minutes.
The cells are then gently resuspended to 1 ml and 1 ~g/ml of
~: biotinylated goat anti-mouse IgG (Southern Biotech, Bi~ngham, Ala) is added.
` The mixture is incubated for 15 Il~inutes on ice and wasbed twice as described~: ~ above with PBS plus l% BS~
: PRBPARATION OF COLUMNS
Carb~xylated Biogel P-30~ (prepared as descnbed above) is allowed
to e~uilibrate to room temperature and placed in a K9/15 column (P.harmacia,
Piscataway, NJ.) to a total~bed~ height of 1 cm. The column is washed with PBS,
25 followed by washes with PBS plus 5% BS~ This column functions as a "pre-
colu~n." The avidin:column contains avidin-conjugated Biogel P-60~, which is
prepared~ as described :above. The avidin-conjugated Biogel is allowed to
equilibrate to room temperature and placed in a K9/15 column to a total bed
height of 4 cm. The column îs then washed with several volumes of PBS, followed
30 by washes with PBS pIus 5% BSA.
IMMuNoADsoRprloN O~ CELLS
~ ~ Cells which have been prepared as descr~bed above are resuspended
: ~ in PBS plus 5% BSA to a volume of 1 ml. The cells are then gently transferred
::
35 onto the top of the gel bed of the pre-column ~ïlter. The cells ~re allowed to filter
through the pre-column and are washed with 1 ml PBS plus 5~ BSA. A
peristaltic pump (Cole-Parmer, r~ock~ord, Ill.~ controls flow from the avidin
WO 93/~8269 PCr/US92/09~24
2 1 2 2 ~ ~3 8 r
æ
column to a rate of abou~ 1 ~al/~ute. Once the cells have almost run down to
the top of the avidin column bedS 1-2 ml of PBS p~us 5% BSA is added to the top
of the avidin column in order to wash out remaining cells. The columD is washed
out with 4-6 ml of PBS plus 5% BSA, followed by 4-6 ml of PBS.
S ..
lRBMOV~L OP ADSORB8D ~llS FR(~ THE~ A~DIN COLUMN
The avidin col~ is placed on top of a 15 ml centrifuge tube. The
valve of the column is opened and 15 ml of RPMI 1 is added to the column with a
wide bore, 9-inch transfer pipette. The RPMI is added to the column while the
10 pipette is used or mecbaI~cally agitating aIld resuspexldi~g the cell bed, thus
allo~g cells to become detached from the gel matrix, and to filter into the
eentrifuge tube. ~e tube is then centrifuged at 400 x g for 5 minutes and
resuspended in cell culture media as described below.
XA~PLE~ 4
Prefere~tial Emichment of Fetal Nucleated Elythroid Cells
ENRICHM~Nr OP FETAL CEL~; W1TH ER~H~OPOIEI1N
(:ells which are separated from the Avidirl-Biogel column above, are
20 resuspended iII Iscoves' Modiiïed Dulbecco's Medium (IMDM) (Gibco, Grand
d, N.'Y.~ containing a fin~ centration of 20% fetal bovine serum (FBS)
(~CLONEn', Logan, Utah), and 2 U/ml of highly purihed recombinant
eryt~opoie~ (Tersy Fox Iabora~ory, Vancou~er~ B.C., Ganada~. Cells are
diluted to S x 106 ~ucleated cells/ml and 200 ~l is plated into each well of a 96
2S well tissue culture plate w~th round bottorns ~Corning Glass Works" CorniIlg, N.Y.).
EN~CHM~Nr OF FE~rAL C~, BY AMMONIUM ION D~eRENrlATION
Cells which are separated from the Avidin column above are
30 ad3usted to a concentra~ion of less than 2 x 107 nucleated cells/~ . One volume of
the cell rnLx~ure is chilled, arld a solution at 29C containing 18 volumes of 0.1844
M NH4Cl and 2 volurnes of 10 ~M acetazolamide is added. After 2 n~inutes, 2
volumes of 3mM NH4HCO3 is rapidly added, and the whole mixture gently
stirred for 3 minutes. Cells are washed several times with PBS by centrifugation in
35 order to remove cellular debris.
WC~ 93/08269 2 1 2 2 1 3 8 Pcr/US92/09024
23
~k~
In Si~u ~yb~idiza~on
E~ched cells are exposed to a hypotonic solution ~.075 M KCl) for
S 12 ~utes at 3rc. The tub~s are inverted once d~g the incubation to keep the
cells suspended. Twen~ drop~ of freshly prepared fixative (3:1 meth~ol:acetic
acid) is added to the cells, vortexed, and then centrifuged for 8 minutes at
appro~mately 250 x g. Fresh fixative is added to the cells, followed by incubation
for one hour at room temperature. The cells are centrifuged for 8 minutes at
10 approximately 250 x g. Fresh f;xative is added and the process is repeated one
more time. Finally, the cells are resuspended in a small amount of fixative and
placed at 4(: ~rernight.
The next day the cells are vortexed and placed onto microscope
slides (Baxter, McGaw Park, 111.) which have been cleaned ~th etha~ol and
15 dipped i~ distilled water. ~e slides are allowed to dly for two days at room
temperature.
The slides a~e beated in 70% ~ormamide in 2 x S~ (0O30 M NaCl,
Q030 M Na c~rate) to 68C-70C ~or 2.5 min~tes. l'he slides are then imme~iatelyplaced in: a ri~se of 70% ethanol in water. Following the r~llse, th~ slides are20 plaoed successively in; 7û~, 95%, aIld 100% ethanol solutions for 5 n~inutes each.
Each alcohol solution mllst be maiIltained at ~ -2~(:~. The sl~des a~e air dried.
A probe is prepared from plas~d DNA according to the method of
Page et al ("Single copy~ sequence hybridizes to polyn~rphic and homologous locion human X and~Y: ch~omosomes," PNAS 79:5352-5356 (1982), from American
:: 25: Type ul~ e Collec~io~ ~(ATCC) No~ 57261; e~cept that biotin-dA~ is
incolporated into the probe. Twent~ ve microliters of the bioti~yla~ed probe (5
~egjml) per slide is placed in a microfuge ~ube. The probe is heated to 7QC for S
minutes the:n immedia~èly placed on ice Twenty microlite~ of the probe solution
, ` is placed o~to e~ch slide and covered with a 22 x 40 mm coverslip. 'rhe slides a~e
30 placed into a box wî~h a wet paper tnweling liner and incubated at 37C for 12-18
hours.
A 50% solution of formamide in 1 x SCC is wanned to 37C.
C~verslips are removed fro~ the slides and immersed into the 50% ~ormamide
solution for 30 minutes. The slides are then placed in 2 x SCC solution for 30
35 minutes with gentle rocking, then in 1 x SCC for 30 minutes with gentle rocking.
FluoresceiIlated avidin (Ve:ctor, Burlingame, Calif.) is diluted 1:1000 (1 l~g/ml~.
The back of the slide and around cell area are w~ped. Two hundred microliters of
WO 93/08269 Pcr/u~92/09024
2122138 ~`
24
the aYidin-fluorescein is added to each slide and incubated in the box for 30
~utes at room temperature. The slides are rinsed sequentia~ly in (1) 4 x SCC
~or 10 minutes with rocl~g, (2) 4 x SCC, 0.1~o Tween-20 for 10 m~mltes ~ithout
rocking, and (33 4 x SSC for 10 ~utes with rocking. The ~ack of ~he slide aIld
5 ar~und cell a~ea is wiped o~. Ten microliters of anti-fade plus propidium iodine
~10 ml PBS, 100 mg p-Phenylene di~e, 90 ml glyceroL pH 8.0, 10 ~g/ml
propidium iodide) is added. The slides are covered with coverslips and stored at4C The slides may be stored ~or several days if necessaly. Target DNA may be
observed under a microscope by the presence of fluorescence.
EXAMPLE 6
Chromosomal Typing
The enriched cells are exposed to 1 ~g of colcemid (Sigma,
15 St. Louis, Mo.) for one hour at 37C. A hypotonic solution (0.075 M KCl) is added
to the cells and incubated ~or 12 minutes at 37C. The hlbes are inverted once
during the incubation to keep the cells suspended. Twen~ drops of freshly
prepared fixative (3:1 metha~ol:acetic acid) is added to the cells, v~r~exed, and
the~ cent~ifuged for B minutes at approximately 250 x g. Fresh ~;xative is added to
20 the cells, followed by incubation for 1 hour at room temperature. The cells are
ce~tri~uged for 8 ~utes at approximately 250 x g and fresh fixative is added
again. This process is repeated one more time. Finally, the cells are resuspended
in a small amou~t of fixative and placed at 4C o~ernight. The next day the cells
e vorte3~ed and placed onto microscope slides ~Ba~ner, McGaw Park, nL) which
25 have bee~ cle~ ed with e~ha~ol ~ d dipped in distilled water. The slides are
allowed to air dry ~or about 34 days, ia~d then are treated with 0.005% ~Iypsin
(Diico ~actot~psirl, VWR Scientific, San Franasco, Calif.) ~r 30 to 35 secondsi.The slides iare washçd ~vice in PBS Plus 1~o FBS7 followed by wiasihiIlg in PBS
vnly. The cells are stained w~th Wright's solution (Sigma Chernical Co., St. Louis,
30 Mo.), followed by t-vo washes with deionized water. The slides are scaDned for
evidence of metaphasie cells and ~ped by conventional cytogenetics.
wo g3/08269 2 1 2 2 1 3 8 P~r/uss2/o9o24
- Culb~re Without Sep~ation
~ ~aATQ~NALBLOOD
S Maternal samples were diluted 1:1 with Phospbate Buffered Saline
(PBS). TweIl~y milliliters were placed in a celltriuge tu~e, foll~wed by 8 ml of
Ficoll ~Iypaque. ~e tube wa~ ce~trifuge for 15 ~utes at 1700 rpm ~SOQxg).
The pelleted cells were washed twice by resuspeIlding and then centrifuging the.cells. ~e cells were resuspended in 20 ml of IMDM culture media eo~t~ng
10 20% Fetal Bovine Serum ~HYCLONE, Logan, Utah), 2mM glut~e, lmM
sodium pyruvate, 0.1 mM NEAA (non-essential amino acids; Whittaker
Bioproducts, Walkersville, Md.)7 and 1 U/ml EPO ~Terry Fox Laboratory?
Vancouver, B.C., Ca~ada. Ten milliliters of the cell suspensiorl was placed intoeach of tWQ T'75 Costar 1asks, and incubated overnight at 37C in a 5% CO2
15 in~ubator. Adherent celIs such as ~lbroblasts are thus removed prior to
separation.
EXA~
:: Separation of Fetal Progenitor Cells on an Affinity Column
20~
Samples from four patients were treated as described above in
Example 7. ~er incubation ove~ight, the eell culture was counted and
: ~ centri~uges 10: minutes ~t 1200 rpm. The supernatant was removed, and the cells
wer~ resuspe~ded in 10 ~ of PBS containing 1~ BSA (Bov~ne Serum Albumin~
5 and centrifuged once more ~for 10 minutes at 1200 rpm (250 x g). The supernatant
was removed and the cells~were resuspended in 1 ml of PBS containing 0.1~o BS~
Antibody 12.8 ~(CellPro, Bothell, Wash.) (an anti-CD 34 antibody~
was added to the cell suspension to a final concentration of 40 llg/ml. The cells
were then iIlcubated for 30 minutes on ice.
30 ~ ~er 30 minutes, the cells were washed ~wice by ce~trifug~tion arldresusper~sio:ll in PBS/1% BS~ Next, biotinyl~ed }abbit anti-mouse IgM antisera
(Zymed Laborat~ries, South San Francisco9 Cal.) was added to a final
concentra$ion of 1~ 0 and~ incubated for 30 minutes on iceO The cells were
washed twice by centrifugation aIld resuspension in PBSl1% BSA, and ~nally
35 res~spended in 1 ml of PB~ containing 5% BS~
An avidinated gel was prepared essentially as described il~ Examples
1 and 2 above, and placed into a scft column (see pendiIlg application U.S. Serial
WO 93/~826~ 2 1 2 2 1 ~ 8 P~r/uss2/o9o24
26
No. 07/5gg,796, which is hereby inco~porated by reference) to a bed depth of 4
cm.
~2
S Culture Post Separa~don
~ dsorbed ~ells which were sepa~ated by the above procedure were
resuspended in IMDM containi~g 20% FBS and 2 U/ml EPO. The cells were.
then placed in 9~ well Costar plates in a volume of 200 ~1 per well ~approximately
10 one million cells per well), and ~ncubated for 5 days at 37C in a 5% C02
incubator.
PR~PARATION OF CELLS FOR MErAPHAS~ SPR~ADS
After incubating for five days, the cells were transferred into
15 microcerltrifuge tubcs and exposed to Colcemid (1 ag in 300 ~1) for one hour at
37C. I'he cells were then:centrifuged for 5 minutes a~ tO00 rpm, and 1 ml of
0.~75M 1~(:1 was added. ~ Twenty drops of freshly prepared fixative (3:1
: Methanol:Acetic Acid) was added to ~he cell suspen~sion, and the cells were
ceotlif iged again for ~ minutes at 10~0 Ipm. OIIe ~ ~f f;xative was added a~d
20 the ~c~lls were left at room temperature ~r 1 hour. T~e cells were then
Centrifu~ed for S minutes at 1000 ~pm, and washed three times in 1 ml of fixative,
~d left in~ 500 ~l overnight at 4~C. The next d~y metaphase spreads were
:prepa~ed.
B. M~HASE S~R~DS~ (KarOtYPIng)
The spreads were allowed to d~y seve~al days at room tempera~ure.
Next, the slides were placed::~in Dif~o Ba~tot~psin~ for one minute (6 drops/45 ml
: PBS). ~e slides were ~insed in PBS containillg 1% ~S, and then in PBS only.
The slides were placed in fresh Wright's stain for 35 seconds, rinsed ~wice, and3~ scaDned under the microscope.
~: : EXAMPLE 1Q
Determination of C~FCs
One ml per 35 rnm plate of Iscove's Methylcellulose (Terry Fox
Laboratories, Ya~couver, British Columbia, Canada) supplemgnted with ~ mM L-
glutamine and 50 ~g/ml gentamic.n was wa~med to 37C. Cells were plated in
WO 93~08~9 2 1 2 2 1 ~ ~ Pcr/vs92/oso24
27
triplicate at 3-~old dilutions to improve the accuracy of the assay. The highestnumber of cells plated was 10S/plate ex~ept for colun~-purified cells wbich wereplated at 3 x 103 and less. The cells were spread evenly over the surface of each
plate and then incubated in a humidified ~ncubator at 3rC with 55'o CO2 in air for
S 10 to 14 days. Color~ies were counted if they contained more than 50 cells andscored as GFU-GM, BFIJ-E, or other (~g., CFU-GEMM). The mlmber of
various ~pes of colonies were summed to E~ive the total number ~f colony-formingcells (~C).
~e results are summarized briefly in Table 1 below:
TABLE 1
CFC Number
,
lS Pregnant Female
Starting cells 8796
Adsorbed cells 613
Unadsorbed cells 1234
Non-pregnant female
: Starling cells 319
~ ,
Adsorbed cells 90
Unadsorbed cells 79
Male
~; :: Starting cells 618
:Adsorbed cells l9S
Unadsorbed cells
When~ CFCs are: compared, it is evident that a significantly higher
number of cells were obtained from the pregnant female, as compared to tbe non-
30 Pregnant female or the male. Although it is possible that the increase in numberis due to mobilization of progenitor cells from the pregnant female's bone
~: marrow, at least a portion of the increase in progenitor cells is due to fetal
progenitor eells in the mother's circulation.
WO 93/08269 P~/U~92Jû9024
.~,,
2122138 28
~k~
In S~ ~Iybndiza~on
Fetal cells which had bee~ enriched as des~ibed above in Example
5 8 were subjected to in si~u hybridization utili~ng a co~ercially available Idt(Chromosome ua situ kit S1370, Oncor, Gaithersburg, MD). CFCs n~Lmber was
also de~e~ed as described above iIl Example 10. The results are briefly set
forth below in Table 2.
TABL~2
SELECrED RESULTS OF F ETAI, CELL SEPARATION FROM ~T~3R~AL
BLOOD FO~WED BY CULTURE AND IN 3ITU HYlBR~ ON
YVITH Y-PROBE
' 15
P~TIEN l GESTATION OElL ~ (Mi0iol~) CFC (Per 105 Cells) IN ~ AMNIO
. . WEER;~S START ADS UNADS START ADS UNADS or CVS
A ~ ~9~05 10 20 6~00 3 Male Malo
: B 16 ~011 31 10 1450 1 Male Male
C 16 350.11 45 2 1610 0 O Female
: :
: : ~ E~P~E 12
ACS A~alysis
Fetal cells which were enriched as descr~bed aboYe in Example 8
: ~ were~ submitted to analysis by F~CS. Briefly, ~pproximately 125,0~ puri~ed cells
were di~1ide~ into two tubes. One tube received an IgG control~ and the second
received ~Bend-10 ~n a~ CD 34 antibody) at a final concentration of 20 ~g/ml.
T~e tubes were incubated for 30 mimltes on ice, then washed twice with 4 ml of
1% BSA in PBS.
Both tubes were then treated with a 1:50 dilution of [FTIC-
conjugatedl goat anti-mouse IgG, incubated for 30 minutes on ice, and washed
with 4 ml of 1% BSA in PBS. After the final wash the cells were suspended in 200~1 of PBS and propidium iodide (1 ~g/ml) and analyzed on a FACScan ~Becton
Dickinson).
WO g3/08~69 2 12 2 13 X P~/US92/09024
~9
EXAMPL~ 13
Plucldng and RecultuIing Colonies
IIIdividual fetal cell colonies were sterilely plucked ~om the
5 methylcellulose culture of Exa~ple 10. The colonies were placed into E~ r~O
media, a~d then placed back ints methylcellulose culture to obsene
dif~eren~iation. From ~he single cell, a coloIly of clonal fe~al cells develops.C~onal fetal cells are identical, and thus may be utilized for assays
wherein the ef~ect of multiple samples are to be tested on cells (ie., to establish
lQ whether a compound is carcinogenic). Some of the cells may function as a control,
while other cells may be subjected to the compound. Simila~ly, multiple assays
may be pe~folmed in order to determine the sensitivity of a ~ell to a certain drug.
In addi~ion, the colonies may be screened ~r a desired response, aII~ clones
~ubjected to more detailed analyses.
From the foregoing, it will be appreciated that, although specific
embodiments of the invelltioa have been described herein for ~he purposes of
illustration, va~i~us modiiïcations may be made wi~hout dev~ati~g from the spirit
20 all~ scope of the invention~ Accordingly, the invelltion is no~ limited except as by
the appended claims.
,
