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BACKGROUND OF THE INVENTION
The present invention relates generally to separation offetal erythrocytes
from maternal blood samples. More particularly, the present invention
provides a system and non-invasive method for enriching the population of
nucleated fetal erythrocytes or nucleated fetal red blood cells ("NRBCs")
obtained from maternal blood samples by separating the NRBCs from the
mother's erythrocytes, leukocytes and other blood components. More
specifically, the present invention offers a system and method for
enriching the population of NRBCs from a maternal blood sample which
concentrates the NRBCs by electrophoresis and/or adsorption-filtration or
affinity filtration.
Physicians have long sought to develop non-invasive methods for prenatal
diagnosis because the available methods, amniocentesis and chorionic
villus sampling (CVS) are potentially harmful to the mother and to the
fetus. The rate of miscarriage for pregnant women undergoing amniocentesis
is increased by 0.5-1%, and that figure is slightly higher for CVS.
Because of the inherent risks posed by amniocentesis and CVS, these
procedures are offered primarily to older women, i.e., those over 35 years
of age, who have a statistically greater probability of bearing children
with congenital defects.
Some non-invasive methods have already been developed to diagnose specific
congenital defects. For example, maternal serum alpha-fetoprotein, and
levels of unconjugated estriol and human chorionic gonadotropin can be
used to identify a proportion of fetuses with Downs syndrome. Similarly,
ultrasonography is used to determine congenital defects involving neural
tube defects and limb abnormalities.
Separation of nucleated fetal erythrocytes from maternal blood has been
proposed as a viable method for facilitating prenatal diagnosis of genetic
disorders. Fetal NRBCs have been separated from maternal blood by flow
cytometry using a lysing reagent (European Published Patent Application
No. 582736, published Feb. 16, 1994); by triple gradient discontinuous
gradient gel electrophoresis (Bhat, et al, U.S. Pat. No. 5,275,933, issued
Jan. 4, 1994); by separation from nucleated cells using leukocyte
depletion and lysis of non-nucleated erythrocytes using ammonium chloride
(Goldbard, PCT Publication WO 9417209, published Aug. 4, 1994); by use of
anti-CD71 monoclonal antibody and magnetic beads and in-situ fluorescence
hybridization (FISH) (Ahlert, et al, German Published patent application
No. 4222573, published Aug. 12, 1993) or by other antibodies specific to a
fetal erythrocyte antigen (Bianchi, PCT Publication Wo 9107660, published
May 30, 1991).
SUMMARY OF THE INVENTION
The present invention demonstrates that fetal nucleated red blood cells
exhibit consistent migration patterns in an electric field according to
surface charge density which are different and distinct from the migration
patterns of adult enucleated red blood cells. By using a novel charge-flow
separation (CFS) method, described hereinafter, the present invention is
able to divide maternal blood into fractions according to the surface
charge density characteristics of each cell type. As the blood cells in a
maternal blood sample move through the CFS apparatus, they are focused
into compartments by opposing forces, namely buffer counterflow and
electric field, and then are directed into waiting collection tubes. An
apparatus and method of counterflow focusing suitable for use with the
system and method of the charge-flow separation of the present invention
are disclosed in our U.S. Pat. Nos. 5,336,387 and 5,173,164, which are
hereby incorporated by reference. A preferred embodiment of a CFS
apparatus and method will be described in greater detail hereinafter.
The inventive CFS system and method has been successfully used to recover
nucleated red blood cells from the peripheral circulation of pregnant
women. The recovered NRBCs were identified histologically. The NRBCs
exhibited consistent migration patterns whether they came from maternal
blood or from umbilical cord blood collected at birth. No NRBCs were found
in blood from nulliparous women.
Because the inventive system and method of charge-flow separation of NRBCs
from maternal blood is based on the intrinsic physical properties of the
NRBCs, there is little need for extensive preparation of the maternal
blood sample. The cells may be processed at greater than or equal to
60,000 cells per second and specialized training is not required. When the
inventive charge-flow separation system and method is used, the recovered
cells are viable, thus raising the possibility of further enrichment by
cell culture.
In conjunction with or in addition to the charge-flow separation system and
method, the present invention also includes an affinity separation method
for separating NRBCs from other cell populations in a maternal blood
sample. The adsorption-filtration affinity method of the present invention
entails layering a maternal blood sample onto a fibrous
adsorption-filtration filter medium having a nominal pore size of about 8
microns and being capable of 40-80% leukocyte immobilization, with a
70-80% post-wash leukocyte retention rate, and which is extremely
hydrophilic, being capable of wetting with solutions having surface
tensions of up to 85-90 dynes, which has a hold up volume of 40-70
.mu.l/cm.sup.2 for a single layer of adsorption-filtration filter medium
and which is characterized by low to medium protein binding. The preferred
adsorption-filtration separation filter medium is that sold by Pall
Corporation under the trademarks "LEUKOSORB" TYPES A and B or that
described in U.S. Pat. Nos. 4,923,620, 4,925,572, or European Patent No.
313348, each of which is hereby incorporated by reference.
The charge-flow separation system and method and the adsorption-filtration
separation system and method of the present invention may be used
separately or may be used in conjunction with one another to achieve
enrichment of the nucleated fetal red blood cell population in a maternal
blood sample.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a diagrammatic view of a first embodiment of the charge-flow
separation apparatus according to the present invention.
FIG. 2 is a perspective view of a separation cell according to the present
invention.
FIG. 3 is a cross-sectional view taken along line 3--3 of FIG. 2.
FIG. 4 is a cross-sectional view illustrating multiple adjacent separation
cells according to the present invention.
FIG. 5 is a diagrammatic view of a second embodiment of the charge-flow
separation apparatus according to the present invention.
FIG. 5A is a perspective view of an end lateral buffer flow cell in
accordance with a second embodiment of the charge-flow separation
apparatus according to the present invention.
FIG. 6 is a flow diagram illustrating a first embodiment of the
adsorption-filtration separation method of the present invention.
FIG. 7 is a flow diagram illustrating a second embodiment of the
adsorption-filtration separation method of the present invention.
FIG. 8 is a flow diagram illustrating a third embodiment of the
adsorption-filtration separation method of the present invention.
FIG. 9 is a flow diagram illustrating a fourth embodiment of the
adsorption-filtration separation method of the present invention operated
in conjunction with the charge-flow separation method of the present
invention.
DESCRIPTION OF PREFERRED EMBODIMENTS
FIRST EMBODIMENT
Charge-flow Separator
Turning to the accompanying Figures, and with particular reference to FIGS.
1-4, there is shown a multi-compartment charge-flow separator 10 according
to a preferred embodiment of the present invention. The charge-flow
separator is comprised of an array of subcompartments 12 with two
electrode compartments 30, 32 on either end of the array. All
subcompartments 12 for this electrophoretic separation cell are identical.
Each subcompartment 12 consists generally of a planar body which has a
narrow channel 16 formed within the planar body. Channel 16 acts as the
separation chamber for processing the sample. The subcompartments 12 are
adjacently arrayed in a co-planar fashion. Membranes 17 are interdisposed
between the individual subcompartments 12. Membranes 17 which serve as
delimiting inter-subcompartmental boundaries for the narrow channel 16 and
effectively separate each adjacent channel 16 into discrete separation
chambers. Each subcompartment 12 is provided with a linear array of
openings 20 which receive a bolt or other fastening means to clamp
together the array of aligned subcompartments 12. Each subcompartment also
is provided with a linear array of alignment openings 22, which receive
alignment pins to maintain a uniform width of the channel 16 along its
longitudinal aspect. The array is bolted or clamped together, whereby the
electrode compartments function as end-plates which provide a rigid
structural support. With the electrode compartments 30, 32 provided on
either end of the array, an electrical field applied to the
electrophoretic separator runs perpendicular to the planar aspect of
subcompartments 23 and the membranes 17. Appropriate electrolytes are
pumped into their respective electrode compartments. Continuous recycling
of the electrolytes removes electrolytic gases.
The membranes 17 reduce inter-subcompartmental fluid convection and mixing
of the separated sample zones. A parallel array of membranes 17, oriented
perpendicular to the separation axis, confines fluid convection to the
individual subcompartments 12. The parallel array of membranes 17 benefit
the separation process while preventing detrimental fluid convection
between subcompartments 12.
Each subcompartment 12 is provided with a heat exchanger 14 to dissipate
Joule heat generated during the electrophoretic separation. The heat
exchanger 14 is disposed in the channel 16 of each subcompartment 12 such
that at least a substantial portion of the longitudinal inner walls of
channel 16 formed by the planar body 12, are associated with heat
exchanger 14. According to the preferred embodiment of the invention, heat
exchanger 14 comprises either a quadrilateral or circular cross-sectional
tubing having a depth or diameter corresponding to the thickness of the
planar body 12. The heat exchanger 14 connects to inlet and outlet ports
18 at the ends of the length of the cavity. Inlet and outlet ports 18 are
preferably formed by providing co-axially aligned inlet and outlet ports
18 in adjacent arrayed subcompartments 12, each port 18 having fluid
conduit 19 between the port 18 and the heat exchanger 14. Fluid conduit 19
may be integral with heat exchanger 14 or a discrete component.
Sample inlet 11 and collection 13 ports are also provided at each end of
channel 16 and are in fluid flow communication therewith. Both inlet port
11 and collection port 13 are connected to a multichannel pump 60 which
pumps the sample solution into channels 16 of individual subcompartments
12 and concurrently removes separated solution from the collection ports
13.
The volume of the electrophoretic separator according to the present
invention is determined by the number of subcompartments 12 and the length
of channel 16 in each subcompartment 12.
According to the best mode contemplated for the invention, each
subcompartment 12 is preferably 0.1 to 0.4 cm in thickness, and may be die
cut, extruded, molded or otherwise formed as may be known in the art.
Regardless of the length of channel 16 or number of subcompartments 12,
the thickness of the each subcompartment 12, and, hence, channel 16, i.e.,
the distance between two membranes 17, should be about 0.1 to about 0.4
cm. Further, an effective dimension of the channel 16 which forms the
separation chamber, should be about 50 cm in length, 0.2 cm in width and
0.3 cm in thickness, which yields an effective total volume of 3 ml within
channel 16.
Because of its dielectric properties, a silicone rubber material is well
suited for sample containment in an electrophoretic separator, however,
other electrically insulating materials are also effective. Because of its
elastic properties, silicone rubber, together with the membranes 17, acts
as a gasket between adjacent subcompartments 12 when the subcompartment
array (FIG. 1) is bolted or clamped together.
The membranes used in the embodiment of the present invention are
preferably woven polytetrafluoroetylene, polyvinylidone fluoride (FVDP),
cellulose, nitrocellulose, polycarbonate, polysulfone, microporous glass
or ceramics, or woven monofilament nylon screens. The screens may be
formed of a woven filament, or may be microporous non-woven materials, or
solid matrices with opening formed by sacrificial sites imparted by
irradiation by laser or other energy sources such as gamma irradiation.
The electrode compartments 30, 32, provided at each end of the
subcompartment array, provide a cavity for a platinum electrode. The
electrode compartments 30, 32 are constructed from a suitable plastic
material, preferably acrylic, and serve as a rigid end plate allowing the
subcompartment array to be bolted together. In order to remove
electrolytic gases from the electrolytic cavity, they are equipped with
inlet and exit ports for the flow-through operation of the electrolytes.
The electrolytes may be recirculated within each compartment 30 or 32, may
be flowed through each compartment 30, 32 in a single pass or the anolyte
may be mixed with the catholyte.
To facilitate assembly of the apparatus 10, the subcompartments 12, and the
electrode compartments 30, 32, have a plurality of openings perpendicular
to their flat faces. Bolts are inserted into the openings, which form one
long bore hole upon the co-axial alignment of the components, which
tightened to seal the subcompartments and electrode compartments against
leakage. Additionally, a plurality of alignment openings 22 are provided
in the planar bodies 12 and adjacent to or in close proximity to the
channel 16. The alignment openings 22 serve to maintain uniform
longitudinal alignment of channel 16, by engagement upon alignment pins
(not shown).
Sample inlet 11 and collection 13 ports may be disposed within the silicone
rubber material and pass from each end of the channel 16 and extend
external to the planar body of the subcompartment 12. Each of the sample
inlet 11 and collection 13 ports are connected to the multichannel pump
60.
Continuous-flow processing requires the provision of means for dissipating
the Joule heat generated by the applied electric field. The apparatus of
the present invention has the heat exchange means 14 for the recirculation
of a coolant either attached to, or made as an integral part of the planar
body of each subcompartment 12 such that the heat exchange means 14 forms
side walls of channel 16. The heat exchange means 14 according to the
preferred embodiment of the invention, consists of cooling tubes 14 having
the same cross-sectional diameter or depth as that of the channel 16.
Except as hereinafter described otherwise, each subcompartment 12 has
associated with it a parallel pair of cooling tubes 14, each of which has
inlet and exit ports 18 provided at the ends of the longitudinal axis of
the subcompartment 12. The inlet and outlet ports 18 of all
subcompartments 12 open into the same feed lines running perpendicular to
the planar aspect of each subcompartment 12 through the array. The feed
lines on either end of the silicone rubber spacer are formed by openings
perpendicular to the planar aspect of the planar body of each
subcompartment 12 and are in fluid flow communication upon bolting or
clamping of the subcompartment array. The coolant is supplied from a
coolant supply 50 and is continuously recirculated by a pump 52.
The cooling tubes 14 may be fashioned of any tubular structure which is
electrically insulated from the applied electrical field. Cooling tubes 14
may be discrete from or integral with the planar body of each
subcompartment 12. For example, cooling tubes 14 may be made of a plastic
material which has a high dielectric strength to avoid electrical
conduction to the coolant and has thin walls to facilitate high thermal
transfer rates. Suitable plastic materials are tetrafluoroethylene or
fluorinated ethylpropylene resins marketed under the trademark TEFLON or
polypropylene co-polymers, polyethelyene or silicone. Alternatively, the
cooling tubes 14 may be made of a ceramic or glass having high thermal
transfer properties, and the glass or ceramic tubes may have metalization
layers on luminal surfaces thereof, which is thereby electrically isolated
from the electric field and which facilitates heat transfer to the cooling
medium. Similarly, cooling tubes 14 which are integral with the planar
body of each subcompartment 12 may consist of chambers within each planar
body for circulating a coolant medium therethrough, or may consist of
inter-subcompartmental channels formed in the planar surface of each
planar body and which reside between adjacent subcompartments 12.
Regardless of the configuration of the cooling tubes 14, they must be in
thermal communication with the separation channel 16 and capable of
conducting Joule heat away from the separation channel 16.
Cooling tubes 14 may have any suitable cross-sectional shape, but are
preferably circular or quadrilateral. Cooling tubes 14 preferably have a
cross-sectional dimension which corresponds to the thickness of the planar
body forming the subcompartment 12, and, therefore, have substantially the
same width or depth as that of the channel 16. Non-integral cooling tubes
14 may be affixed to the side walls of the channel 16 by gluing, welding,
or other suitable method of affixation as may be known in the art, the
cooling tubes may be molded directly into the material forming the planar
body of the subcompartment 12, or the cooling tubes 14 may be extruded or
otherwise formed as an integral part of the planar body of the
subcompartment 12.
Sufficient cooling of the sample volume in the cavity is achieved only when
the entire volume of the sample fluid is in close proximity to the surface
area of the heat exchange means 14. It has been found preferable to have
the sample fluid within a range of about 0.05 cm to about 0.15 cm away
from any heat exchange means 14 surface to provide effective cooling of
the sample fluid. Thus, the desirable width of channel 16, as measured by
the lateral distance between the two parallel cooling tubes 14 is from
about 0.1 cm to about 0.3 cm.
Providing internal cooling adjacent to the separation chamber permits use
of a higher applied potential for the separation process which increases
both the resolution and the speed of the separation. An advantage of the
apparatus according to the present invention over designs of the prior art
is the configuration of a long narrow separation chamber having internal
cooling. As long as the ratio of cooling surface area to process volume
remains constant or is increased, the device can be scaled to any sample
cavity volume desired, simply by increasing the length and the number of
subcompartments 12, without loss of resolution.
SECOND EMBODIMENT
Laterally Introduced Buffer Counterflow Gravity Biased Charge-Flow
Separator
Turning now to FIGS. 5 and 5A, there is illustrated a second embodiment of
a charge-flow separator apparatus 60 which employs a laterally introduced
buffer counterflow and a gravity biased sample flow. The charge-flow
separator apparatus 60 (CFS 60) is largely identical to that of the first
embodiment of the charge-flow separator apparatus 10 (CFS 60), described
above. Specifically, the CFS 60 includes a plurality of planar spacer
members 62 formed in a parallel array, identified as 1-12 in FIG. 5, with
screen members interdisposed therebetween (not shown) as described above
with reference to the CFS 10. Each of the plurality of planar spacer
members 62 are identical to those described above with reference to FIG. 3
and as described in U.S. Pat. Nos. 5,336,387 and 5,173,164, incorporated
herein by reference Two planar end-spacer members 90, identified as 0 and
00 in FIG. 5, form end boundary fluid flow members for introduction and
withdrawal of a buffer counterflow 75 through the parallel array of planar
spacer members 62. Electrodes 61 and 64 form an anode and cathode,
respectively, at opposing ends of the parallel array of planar spacer
members 62 and the planar end-spacer members 90. Electrodes 61 and 64 are
electrically connected to an appropriate variable power supply (now shown)
to provide an electromotive force within the separation chamber. Each of
the electrodes 61 and 64 are in fluid flow communication with an electrode
buffer reservoir 70 and an electrode buffer pump 68 to recirculate an
electrode buffer through the electrode compartments as hereinbefore
described with reference to the CFS 10.
A throughput sample flow 63 is introduced into the parallel array of planar
spacer members 62 from a sample container 58 under the influence of a
throughput pump (T-Pump) 72. The T-Pump 72 is capable of introducing the
throughput sample flow 63 into a first end of a selected one or more of
the planar spacer members 62. A separated sample flow 65 is withdrawn from
a second end of each of the planar spacer members 62 under the influence
of a fraction pump (F-Pump) 73 which feeds the separated samples from each
of the plurality of planar spacer members into a fraction collector 74.
The operational parameters of sample flow 63 and separated sample flow 65,
T-Pump 72, F-Pump 73, and fraction collector 74 are more full set forth
above with reference to FIGS. 1-4 and in U.S. Pat. Nos. 5,336,387 and
5,173,164, incorporated herein by reference.
Two significant areas of difference exist between CFS 60 and CFS 10. The
first of these differences results from the CFS 60 being oriented such
that sample inflow 63 occurs at the bottom each of a plurality of
separation chambers 62 and separated sample outflow 65 occurs at the top
of each of the plurality of separation chambers 62. In this manner, the
sample flow through the plurality of separation chambers 62 is biased
against ambient gravity 85. Biasing the sample flow through the plurality
of separation chambers 62 reduces zone sedimentation effects on cell
populations normally found in sample flows which are normal to the gravity
vector 85. By reducing the zone sedimentation effect on the cell
populations, the gravity biasing of the present invention effectively
increases throughput with correspondingly longer residence times in the
separation chamber.
The second of these differences is a lateral orientation of the buffer
fluid flow 77 and 79. As best illustrated in FIG. 5A, the lateral buffer
fluid flow 77 and 79 is facilitated by modification of the spacer members
12 described above with reference to FIG. 3. Turning to FIG. 5A, a
generally rectilinear planar end-spacer member 90 is provided with a fluid
flow opening 96 longitudinally oriented therein. The fluid flow opening 96
is bounded on an upper, a lower and lateral surfaces thereof by the spacer
member 90, but is open to frontal and rearward planar surfaces of the
planar end-spacer member 90. A cooling member 94, similar to the cooling
member 14 described above with reference to the CFS 10 is disposed along
one lateral surface of the fluid flow opening 96 forming one lateral
boundary of the fluid flow opening. The cooling member 94 is connected, in
fluid flow communication with inlet and outlet ports 98 also disposed in
the planar end-spacer member 90. Inlet and outlet ports 98, in turn,
communicate with an external fluid cooling medium and heat exchanger (not
shown, but identical to that of the CFS 10, heretofore described).
A plurality of buffer ports 93 are disposed on a second lateral surface of
the fluid flow opening 96, which open into the fluid opening 96 in the
direction of and opposite to the cooling member 94. Each of the plurality
of buffer ports 93 are connected, through a first lateral wall 95 of the
planar end-spacer member 90, to a plurality of tubular members 91. Tubular
members 91 serve either to convey a buffer fluid flow 77 from an external
buffer reservoir 80 to the plurality of buffer ports 93 under the
influence of a multichannel counterflow buffer pump (C-Pump) 76 or from
the plurality of buffer ports 93 to either a second fraction collector or
to waste 82 under the influence of a second multichannel fraction pump
(F-Pump') 78. Thus planar end-spacer 0 serves to receive the buffer inlet
flow 77 from the C-Pump 76, while planar end-spacer 00 serves to convey
the buffer outlet flow 79 to either fraction collector or waste 82 under
the influence of F-Pump' 78, thereby creating the buffer counterflow 75
across the parallel array of planar spacer members 62 in a direction
opposing the applied electrical field 71.
The buffer inlet flow 77 and the buffer outlet flow 79 are, therefore,
laterally oriented relative to the planar end-spacer member 90 used as the
buffer inlet member and the fluid opening 96 within the planar end-spacer
member 90. By orienting the buffer ports 93 laterally relative to the
planar end-spacer member 90, the buffer inlet flow 77 and the buffer
outlet flow 79 are perpendicular to the axis of the buffer counterflow 75
through the plurality of planar spacer members 62 and the axis of the
applied electrical field 71. In this manner a generally laminar buffer
counterflow 75 through the plurality of planer space | | |
