WO2011028483A2 - Procédés et appareil pour la ségrégation de particules, comprenant des sources supplémentaires de collecte de prélèvements pour la séparation de cellules foetales nucléées - Google Patents

Procédés et appareil pour la ségrégation de particules, comprenant des sources supplémentaires de collecte de prélèvements pour la séparation de cellules foetales nucléées Download PDF

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Publication number
WO2011028483A2
WO2011028483A2 PCT/US2010/046350 US2010046350W WO2011028483A2 WO 2011028483 A2 WO2011028483 A2 WO 2011028483A2 US 2010046350 W US2010046350 W US 2010046350W WO 2011028483 A2 WO2011028483 A2 WO 2011028483A2
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WIPO (PCT)
Prior art keywords
passage
fluid
cells
cover
particles
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Application number
PCT/US2010/046350
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English (en)
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WO2011028483A3 (fr
Inventor
George Hvichia
David Counts
Gary Evans
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Parsortix, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Parsortix, Inc. filed Critical Parsortix, Inc.
Priority to US12/910,299 priority Critical patent/US20110065181A1/en
Publication of WO2011028483A2 publication Critical patent/WO2011028483A2/fr
Publication of WO2011028483A3 publication Critical patent/WO2011028483A3/fr
Priority to US15/586,981 priority patent/US20170234851A1/en
Priority to US15/870,381 priority patent/US20180299425A1/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5002—Partitioning blood components

Definitions

  • cord blood blood drawn from the umbilicus shortly after delivery
  • cord blood is a rich source of stem cells, such as embryonic stem cells and hematopoietic stem cells.
  • Hematopoietic stem cells are useful for treating blood disorders.
  • Methods of storing cord blood samples are known. These methods have the drawback that a relatively large volume (e.g., 100 to 250 milliliters) of blood must be stored in order to preserve a sufficient number of stem cells for use in future medical procedures.
  • the large volume of cord blood that is stored increases the cost and decreases the convenience of the procedure.
  • the stored volume could be decreased significantly (e.g., to 0.1 to 1 milliliter) if stem cells could be readily separated from cord blood prior to storage.
  • present methods of separating stem cells from cord blood are expensive, cumbersome, and sometimes ineffective. There is a need for an efficient and cost-effective method of segregating stem cells from cord blood.
  • cells of apparently fetal origin can be found in the blood of pregnant women and in the blood of women who have previously been pregnant. These cells can have male DNA when the mother has given birth to or is pregnant with a male child, and therefore the DNA appears to originate from the fetus. These cells are rare in the maternal bloodstream; there may be only 10 to 12 cells per milliliter of maternal blood. Among fetal-like cells observed in maternal blood, fetal trophoblasts can degrade relatively quickly after the woman gives birth. Other kinds of fetal-like cells have been reported to endure in the blood of women for years or decades following pregnancy albeit in small numbers.
  • the subject matter disclosed herein can be used to segregate and manipulate biological cells, organelles, and other particles from mixed populations of particles or cells.
  • the present disclosure relates to an apparatus for segregating particles such as cells.
  • the apparatus includes a body, a cover, and a separation element.
  • the body and cover define a void.
  • the separation element is contained within the void.
  • the void has a fluid inlet region and a fluid outlet region.
  • the separation element has a shape that defines a stepped passageway that fluidly connects the inlet and outlet regions in the void.
  • the separation element includes a first step and a second step, each of which extends into the stepped passageway.
  • the passage bounded by the second step is narrower than the passage bounded by the first step.
  • fluid can flow from the inlet region, through the first passage, through the second passage, and into the outlet region.
  • Particles suspended in the fluid can transit the first and second passages if the size of the particles does not exceed the narrow dimension of each passage, or if the particles are sufficiently deformable that, in a deformed shape, they can squeeze through each passage.
  • Particles can be segregated by selecting a narrow dimension for the second passage that permits only some of the particles to pass therethrough.
  • the narrow dimension of the first passage can be selected such that particles in the fluid can pass through the first passage individually, but two particles cannot pass through the first passage simultaneously if they are stacked across the narrow dimension of the first passage.
  • the apparatus can include a fluid inlet port for facilitating fluid flow from outside the apparatus into the inlet region, a fluid outlet port for facilitating fluid from the outlet region to the outside of the apparatus, or both.
  • a fluid displacement device e.g., a pump or a gravity- fed fluid reservoir can be fluidly connected with one or both of the inlet and outlet ports to facilitate fluid flow through the stepped passageway.
  • Such flow can be in the direction from the inlet region toward the outlet region, for the purpose of segregating particles.
  • Fluid flow can be in the direction from the outlet region toward the inlet region, for example to flush out particles that were unable to traverse the second passage during inlet region-to-outlet region fluid flow.
  • the steps of the separation element define passages within the stepped passageway, and there can be two or more such steps.
  • the steps can be formed from planar regions that meet at a right angle (forming classical right-angled steps), or the riser portion (i.e. the transitional face) of the step can be inclined, such that a first planar step region can be connected to a second planar step region by a sloped flat surface or by a curved surface.
  • the planar step regions can be substantially parallel to a portion of the cover, a portion of the body, or both, and should have a length (in the direction of bulk fluid flow) equal to a multiple (e.g., 2, 4, 10, or 1000) of the narrow dimension of the passage it bounds.
  • the width of the planar region (in the direction perpendicular to bulk fluid flow) should be equal to a multiple (e.g., 10, 1000, of 10000) of the narrow dimension of the passage it bounds.
  • the apparatus can have one or more supports within the void for maintaining the dimensions of the stepped passageway during assembly and operation of the device.
  • the support can completely span the distance between the separation element the body or the cover or it can span only a portion of that distance, to provide room for deformation of an element (e.g., upon assembly and clamping of the apparatus).
  • the present disclosure includes a method of segregating particles. The method includes introducing particles at the inlet region of the apparatus, permitting them to move (i.e., by endogenous cell motility or under the influence of induced fluid flow) through a stepped passageway to an outlet region. At least some of the particles are prevented from entering the outlet region by a step in the passageway, resulting in segregation of the particles.
  • Particles able to traverse all steps in the stepped passageway can be collected from the outlet region.
  • Particles unable to traverse at least one step in the stepped passageway can be collected from a portion of the passageway upstream from the step that inhibits their movement through the passageway.
  • trapped particles can be recovered by inserting a device (e.g., a catheter) into the stepped passageway, by reversing fluid flow and flushing the trapped cells out of the passageway by way of the inlet region, or by disassembling the device and recovering the trapped particles directly. If the trapped particles are cells, they can be lysed within the stepped passageway and the lysis products collected by flow in either direction.
  • Figure 1 consists of Figures 1A and IB.
  • Figure 1A is an elevated view of a portion of the apparatus in one embodiment.
  • Figure IB is a vertical section of the portion of the apparatus shown in Figure 1A, taken along plane IB, showing a body 10 which defines a void 11.
  • a cover 12 is disposed across the void 11 forming a fluid-tight seal with the body 10.
  • a separation element 14 having a first step 61 and a second step 62 is disposed within the void 1 1 between an inlet port 16 and an outlet port 18.
  • the first step 61 has a broad surface 31 and a transitional face 41.
  • the second step 62 has a broad surface 32 and a transitional face 42.
  • Figure 2 consists of Figures 2A, 2B, and 2C.
  • Figure 2A is an elevated view of a portion of the apparatus in an embodiment having inner support structures 20.
  • Figure 2B is a vertical section of the portion of the apparatus shown in the Figure 2A, taken along plane 2B.
  • Figure 2C is a vertical section of a portion of the apparatus shown in Figure 2A, taken along plane 2C.
  • Figure 3 consists of Figures 3 A and 3B and illustrates a configuration of the apparatus described herein wherein the geometry of the first and second passages can be selected to achieve substantially constant linear flow velocity throughout the first and second passages.
  • Figure 3 A is an elevated view of a series of passages wherein the width of each passage increases in the direction from the inlet region to the outlet region.
  • Figure 3B is a vertical section of the series of passages shown in Figure 3A taken along plane 3B, wherein the height of each passage decreases in the direction from the inlet region to the outlet region.
  • Figure 4 is a perspective view of a portion of a separation element showing the length height "ft", and width "ut” of a step, and indicating the direction of bulk fluid flow "BFF" past the step.
  • Figure 5 is a color image showing an elevated view of the cover 12 of an assembled apparatus, showing the light pattern in an appropriately assembled apparatus, as described herein in Example 2.
  • Figure 6 is a diagram that illustrates the relative arrangements of the cover 12, base 10, and first, second, third, fourth, fifth, sixth, seventh and eighth steps (61 -68) of the separation element 14 of an apparatus used in experiments described herein in Examples 3 and 4.
  • the direction of fluid flow is shown as 'D.'
  • Figure 7 is a map showing the approximate locations within the separation region of the experiments described herein in Example 4 at which fetal-like cells were found.
  • the portion of the Relative Vertical Position designated “Outlet Area” corresponds approximately to the portion of the cassette at which steps having cover-to-step distances of 4.2 and 4.4 micrometers were located, and the portion of the Relative Vertical Position designated “Inlet Area” corresponds approximately to the portion of the cassette at which steps having cover-to-step distances of 5.2 and 5.4 micrometers were located.
  • the disclosure relates to an apparatus for segregating particles on the basis of their ability to traverse a passage.
  • Particles e.g., particles suspended in a liquid or gaseous fluid or particles in a vacuum
  • the stepped passageway contains at least two passages that are fluidly connected in series, each passage having a narrow dimension. Most or all particles in the fluid are able to move into the first passage, but only some of the particles are able to move through the second passage. The net result is that some particles can move through the entire stepped passageway, while other particles are retained within the apparatus, such as within the first passage. Segregation of particles is thus achieved.
  • Movement of particles can be motivated by fluid flow, gravity, vibration, or any combination of these, for example.
  • the "length” of a step refers to the distance that the step extends in the direction of bulk fluid flow through the passage corresponding to the step.
  • the "height" of a step refers to the distance that the step extends in the direction away from the separation element beyond the preceding (i.e., upstream) step surface.
  • the "width” of a step refers to the distance that the step extends in the direction that is perpendicular to bulk fluid flow over the step.
  • the "narrow dimension" of a passage refers to the distance between the broad portion of a step of the separation element and the opposed, generally parallel, face of the apparatus (e.g., the face of the cover or body that faces the void).
  • the narrow dimension of the passage is the length of a line in that plane extending between and at right angles to each of the flat surface of the step and the flat surface of the opposed face of the apparatus.
  • the "narrow dimension" of each of passages 51 and 52 in Fig. IB is the minimum distance between each of the step surfaces 31 and 32 and the nearest surface of cover 12.
  • the "flow area" of a passage is a cross-section of the passage taken in a plane perpendicular to the direction of fluid flow in the passage.
  • the disclosure relates to an apparatus for segregating particles on the basis of their ability to flow through at least two passages, the second (downstream) passage 52 being narrower than the first (upstream) passage 51.
  • the apparatus includes a separation element 14 disposed in a void 1 1 formed by a body 10 and cover 12. Within the void 11, the separation element 14 separates an inlet region 15 of the void from an outlet region 17 of the void. The inlet and outlet regions are in fluid communication by way of a stepped passageway defined by the separation element 14 and one or both of the body 10 and cover 12. Steps formed in the separation element define the first and second passages.
  • the apparatus optionally has an inlet port 16 that fluidly communicates with the inlet region 15 of the void 11 and an outlet port 18 that fluidly communicates with the outlet region 17 of the void 1 1, to facilitate provision and withdrawal of fluid to the inlet and outlet regions.
  • particles in the inlet region 15 pass into the first passage 51 and, if they are able, into the second passage 52. Particles in the second passage 52 pass to the outlet region 17. Cells that are not able to pass into or along the second passage 52 do not reach the outlet region 17. In this way, particles able to reach the outlet region 17 are segregated from particles that are not able to reach the outlet region 17.
  • the two populations of particles can be separately recovered from the apparatus. For example, particles at the outlet region 17 can be recovered in a stream of liquid withdrawn from the outlet region 17 (e.g., by way of an outlet port or by way of a catheter inserted into the outlet region 17.
  • Particles unable to pass through the second passage 52 to the outlet region 17 can be recovered by flushing them, in the reverse direction, through the first passage 51 and into the inlet region 15. Such particles can be withdrawn from the inlet region 15. Alternatively, particles unable to pass through the second passage 52 to the outlet region 17 can be left in the apparatus or recovered by disassembling the apparatus. Particles unable to enter either the first passage 51 or the second passage 52 can be recovered from the inlet region 15.
  • the apparatus described herein can be used in a wide variety of applications.
  • the device can be used in applications in which one or more of the segregated particle populations are identified or further manipulated, for example.
  • the construction and operation of the apparatus resist clogging by the particles being segregated, relative to devices previously used for particle separation.
  • the particles segregated using the apparatus described herein can be suspended in a liquid or gaseous fluid, or in no fluid at all (e.g., in a vacuum).
  • any fluid in which particles are suspended can either be flowed through the apparatus or remain static. That is, particles can be segregated regardless of whether any fluid in which they are suspended is caused to move through the spaces of the apparatus.
  • particles in a mixture of dry particles can be segregated by providing the mixture to the inlet region and vibrating or shaking the device (oriented such that gravity will tend to draw the particles through the separation region).
  • Such use can be beneficial in situations in which suspension of particles in a fluid is considered undesirable or unnecessary (e.g., when separating plant seeds from other particulate matter such as seeds of other plants).
  • the apparatus has a body 10 and a cover 12 defining a void 11 therebetween.
  • the stepped passageway is also defined by a surface of the body 10, a surface of the cover 12, or by a combination of these, that is opposed to the stepped surface(s) 31 and 32 of the separation element 14. (i.e., in an orientation such that the stepped
  • stepped passageway-defining surface(s) of the body 10 and/or cover 12 contact the stepped passageway-defining surface(s) of the separation element 14 in such a way that the surfaces form an extended lumen (i.e., the stepped passageway) between the surfaces.
  • most or all of the stepped passageway-defining surfaces can be formed or machined into a separation element 14 that is an integral part formed in a recess of the cover 12 or the body 10, the recessed portion being surrounded by a flat surface, so that the opposed surface of the body 10 or the cover 12 need only be another flat surface in order to form the stepped passageway upon contact between the flat surfaces of the body 10 and cover 12.
  • the separation element 14 is preferably integral with (formed or machined as a part of) one of the body 10 and the cover 12.
  • the operative portion of the apparatus consists of essentially two pieces— either a cover 12 and a body 10 having a separation element 14 as a part thereof, or a body 10 and a cover 12 having a separation element 14 as a part thereof. It is not important which of the body 10 and cover 12 bears the separation element 14, because the body 10 and cover 12 form the walls of and define the void 1 1 in which the separation element 14 is disposed.
  • a portion of the part not bearing the separation element 14 is simply a flat surface that mates with flat edges of the part bearing the separation element 14 and having the void 11 therein, so that upon assembly of the two parts, the void 1 1 is sealed by mating of the flat surfaces and the separation element 14 is disposed within the thus-sealed void 11.
  • one of the parts has both the void 11 and the separation element 14 formed or machined therein or, alternatively, has the void 1 1 formed or machined therein and has the separation element 14 placed, assembled, formed, or adhered within the void 11.
  • the shapes of the body 10 and cover 12 are not critical, except for the portion(s) of the body 10 and/or cover 12 that define the stepped passageway in the void 1 1 and the portion(s) of the body 10 and cover 12 that mate to seal the void 11.
  • the requirements of the portion(s) of the body 10 and/or cover 12 that define the stepped passageway are discussed in the section of this disclosure pertaining to the stepped passageway.
  • the portion(s) of the body 10 and cover 12 that mate to seal the void 11 do not have any particular shape or location requirements, other than that they should seal the void 1 1 when the apparatus is assembled, with allowances for any orifices (e.g., inlet or outlet ports) that are bounded by both the body 10 and the cover 12.
  • Sealing can be achieved by direct contact between the relevant portions of the body 10 and cover 12.
  • sealants such as adhesives, greases, gaskets, waxes, and the like can be applied on the sealing surfaces of the body 10 and cover 12.
  • the seal should be able to withstand the anticipated internal pressure generated within the apparatus during its operation. For example, in many embodiments, an internal fluid pressure greater than 25 pounds per square inch of gauge pressure (psig) would be unusual, and a seal capable of preventing fluid leaks at this pressure should suffice for such embodiments. More typical operation pressures in embodiments in which biological cells are separated using the apparatus are anticipated to be within the range >0-15 psig.
  • the apparatus can be operated by application of negative (i.e., vacuum) pressure to the outlet region, in which embodiments the seal should prevent the passage of air or liquid from outside the device into the void (other than, of course, by way of the inlet region).
  • negative i.e., vacuum
  • the size and shape of the remaining portions of the body 10 and cover 12 are not critical and can be selected to facilitate, for example, manufacturing, handling, or operation of the apparatus.
  • the body 10 can have the void 1 1 and separation element 14 formed or machined therein, and portions of the body 10 outside the void 1 1 can be formed or machined to adapt the body 10 for securing it in a frame or holder of fixed geometry.
  • the body 10 can have flanges, handles, threaded holes, smooth bores, impressions or indentations for holding a clamp, or other features formed, applied, or machined therein or thereon, and such features can facilitate reproducible orientation of the body 10 in a device for operating the apparatus or reproducible orientation of the body 10 in a device for machining one or both of the void 11 and the separation element 14 in the body 10.
  • the body 10, the cover 12, or both can define a port through which fluid can be introduced into or withdrawn from the void 1 1.
  • the body 10 can define an inlet port 16 that fluidly communicates with the inlet region 15. Fluid introduced into the inlet port 16 can flow into the inlet region 15, displacing fluid already there (because the void is sealed) into the stepped passageway, and thence into the first passage 51 and the second passage 52 and into the outlet region 17. Particles suspended in fluid in one of these regions and passages can be carried into a downstream region or passage, provided the particle can flow through the present and intervening passages and regions.
  • withdrawal of fluid from the outlet region 17 by way of an outlet port 18 formed in the body 10 can induce fluid flow from passages in fluid communication with the outlet region 17 and from passages and regions in fluid communication therewith.
  • Ports can be simple holes which extend through the cover or body, or they can have fixtures (burrs, rings, hubs, or other fittings) associated with them for facilitating connection of a fluid flow device to the port.
  • the body 10, cover 12, or both can define an inlet port 16 in the inlet region 15 of the void 11, an outlet port 18 in the outlet region 17 of the void 1 1, or both an inlet port 16 and an outlet port 18.
  • Fluid can be introduced into the inlet region 15 through the inlet port 16.
  • Fluid can be withdrawn from the outlet region 17 through the outlet port 18.
  • Continuous introduction of fluid into the inlet region 15 and simultaneous withdrawal or emission of fluid from the outlet region 17 can create a continuous flow of fluid through the apparatus.
  • continuous withdrawal of fluid from the outlet region 17 and simultaneous influx or introduction of fluid into the inlet region 15 can create continuous flow.
  • the body 10 and the cover 12 form a void 1 1 when they are assembled.
  • the void 1 1 has an inlet region 15, an outlet region 17, and a separation region interposed between the inlet region 15 and the outlet region 17.
  • a separation element 14 is disposed within the separation region and, together with the body 10, the cover 12, or both, defines a stepped passageway.
  • the stepped passageway includes at least a first passage 51 and a second passage 52, that are fluidly connected in series and that are defined by steps in the separation element 14.
  • the stepped passageway can include any number of additional steps, each of which can define an additional passage in the void.
  • the inlet region 15, the outlet region 17, and the stepped passageway of the void 11 are filled with a fluid.
  • the entire void 11 is filled with fluid during operation.
  • the only fluid path that connects the inlet region 15 and the outlet region 17 is the stepped passageway. Particles present in the inlet region 15 can enter and pass through the first passage 51 of the stepped passageway unless they are excluded by the size (i.e., the narrow dimension) or shape of the first passage 51.
  • Particles present in the first passage 51 can enter the second passage 52 unless they are excluded by the size (i.e., the narrow dimension) or shape of the second passage 52, or unless their movement through the first passage 51 is inhibited by the size (i.e., the narrow dimension) or shape of the first passage 51.
  • Particles present in the second passage 52 can enter the outlet region 17 unless their movement through the second passage 52 is inhibited by the size (i.e., the narrow dimension) or shape of the second passage 52. Movement of particles within the apparatus can be induced by fluid flow through the apparatus, by intrinsic motility of the cells, or a combination of the two. Over time, particles unable to enter the first passage 51 will be segregated in the inlet region 15;
  • particles able to enter the first passage 51 but unable to enter the second passage 52 will be segregated in the first passage 51 ; particles able to enter the second passage 52 but unable to freely move therethrough will be segregated in the second passage 52; and particles able to move through both the first passage 51 and the second passage 52 will be segregated in the outlet region 17 (or in fluid withdrawn or emitted from the outlet region 17).
  • Particles segregated in this manner can be recovered (using any of a variety of known methods, including some described herein) from their respective locations.
  • a catheter can be inserted into a region or passageway (e.g., the inlet region 15 or the first passage 51) of the apparatus, and particles present therein can be withdrawn by inducing suction in lumen of the catheter.
  • backflushing i.e., fluid flow from the outlet region 17 in the direction of the inlet region 15
  • backflushing can be used to collect particles present in one or more of the inlet region 15, the first passage 51, and the second passage 52 in fluid collected, withdrawn, or emitted at the inlet region 15.
  • particles present at the inlet region 15 can be collected by a transverse (relative to bulk fluid flow from the inlet region 15 to the outlet region 17 by way of the stepped passageway) fluid flow across the inlet region 15, using ports provided for this purpose in fluid communication with the inlet region 15.
  • the separation element 14 is a part of the apparatus that has a surface that defines part of the stepped passageway.
  • One or both of the body 10 and the cover 12 define the remaining boundaries of the stepped passageway, which fluidly connects the inlet region 15 and the outlet region 17.
  • the separation element 14 has a shape that includes at least two steps, the steps forming at least one of the boundaries of each of the first passage 51 and the second passage 52.
  • One or both of the body 10 and the cover 12 define the remaining boundaries of the first passage 51 and the second passage 52.
  • the stepped passageway is the orifice through which particles move, fluid flows, or both, during operation of the apparatus.
  • the separation element 14 has a stepped structure, which defines the stepped shape of at least one side of the stepped passageway.
  • the separation element 14 has at least two steps, the first step 61 and the second step 62.
  • the first step 61 defines a boundary of the first passage 51 in the stepped passageway.
  • the second step 62 defines a boundary of the second passage 52, the second passage 52 having a smaller narrow dimension (see, e.g., Figure 2B) than the first passage 51.
  • the first and second passages are fluidly connected in series, the second passage 52 being downstream from the first passage 51 during normal operation of the apparatus. Fluid must flow through each of the first and second passages in the stepped passageway in order to travel from the inlet region 15 to the outlet region 17 when the apparatus is assembled.
  • the separation element 14 is associated with at least one of the body 10 and the cover 12.
  • the separation element 14 can be attached to the surface of the body 10 or the cover 12.
  • the separation element 14 can instead be integral with one of the body 10 or the cover 12, such that when the body 10 and the cover 12 are assembled, the stepped surface(s) of the separation element 14 are brought into opposition with the surface(s) of the body 10 or the cover (12) that form the boundaries of the stepped passageway.
  • the separation element 14 can be a part separate from the cover 12 or the body 10. If the body 10, the cover 12, and the separation element 14 are separate parts, then the parts are preferably dimensioned and shaped such that the separation element 14 is held in place by compression between the cover 12 and the body 10 when the apparatus is assembled.
  • Fluid pressures within the apparatus are exerted on all surfaces contacted by the fluid, and such fluid pressures can induce bending or bulging in deformable materials.
  • external pressure applied to parts of the apparatus in order to secure it in its assembled state e.g., one or more clamps which urge the cover 12 against portions of the body 10) can also induce flexation or bulging in flexible materials that form one or more parts of the apparatus.
  • the second passage 52 defined by the separation element 14 and at least one of the body 10 and the cover 12 is the primary mechanism by which particles are segregated by the apparatus in operation, it is preferable that the narrow dimension of the second passage 52 be carefully maintained relatively constant across the width of the second step 62.
  • the second passage 52 has boundaries defined by the second step 62 of the separation element 14 and by one or both of the body 10 and the cover 12. Clamping the body 10 and the cover 12 together can exert external force on a part which forms a boundary of the second passage 52, thereby tending to induce flexation of the part and narrowing of the narrow dimension of the second passage 52. Such flexation and narrowing can be reduced or eliminated by including one or more supports 20 within the lumen of the second passage 52.
  • a support 20 can be, for example, a rod-shaped extension extending from the surface of the separation element 14 that defines the boundary of the second passage 52 in the direction of the opposed surface of the body 10 or the cover 12.
  • an extension having a rectangular cross-section can extend away from the surface of the body 10 or the cover 12 that defines a boundary of the second passage 52 in the direction of the opposed surface of the separation element 14 can form a support 20.
  • More than one support 20 can be arranged in parallel or in series to form one or more solid or segmented walls, and such supports can define multiple flow paths within the void, the multiple flow paths merging at one or both of their ends.
  • a support 20 can be a discrete part disposed in the lumen of the second passage 52 and substantially or fully spanning the narrow dimension between the opposed surfaces of the separation element 14 and the body 10 or cover 12.
  • the supports 20 can enhance the operability of the apparatus under various operating conditions (e.g., with varying clamping pressures or with varying fluid pressures) and extend the life of the apparatus.
  • Supports 20 can also enhance the particle segregating accuracy of the apparatus by preventing the body 10 or cover 12 from deforming and altering the narrow dimensions of one or more of the first and second passages of the stepped passageway.
  • Supports 20 can also be disposed in the void 11 outside of the first and second passages, and span the height of the void. Such supports 20 can maintain the patency of the void 1 1 outside the first and second passages.
  • a support 20 is not integral with a surface impinged by the support 20, the support 20 can be not attached to the surface, adhered to the surface (e.g., using an adhesive interposed between and binding both the surface and a portion of the support), or fused with the surface.
  • Supports 20 can separate an otherwise unitary fluid flow path into two or more fluid flow paths within the void 1 1 (see, e.g., supports 20 in Figure 2A).
  • the apparatus consists of a flat cover 12, a body 10 having a flat surface that mates with the cover 12 and defining a void 11 having an inlet region 15 and an outlet region 17, and a separation element 14 that includes a first step 61 and a second step 62 and is integral with four supports 20.
  • the height of the supports 20 is equal to the depth of the void 11, such that the upper surfaces of the supports 20 are substantially co-planar with the flat surface of the body 10 (as depicted in Figures 2B and 2C).
  • the cover 12 is assembled against the flat surface of the body 10, the top surfaces of the supports 20 contact the surface of the cover 12 that defines the void 11, thereby preventing clamping pressure (applied to the cover 12 to hold it flush against the flat surface of the body 10) from deforming the cover 12.
  • the bracing provided to the cover 12 by the supports 20 serves to maintain the narrow dimension of the second passage 52 and the narrow dimension of the first passage 51, even when clamping pressure that would otherwise deflect the cover 12 inwardly toward the void is applied to the cover 12. If the cover 12 is fused with or adhered to one or more of supports 20, then the apparatus depicted in Figure 2 can also resist expansion of the narrow dimension of the first passage 51 and the second passage 52 that might otherwise result from outward (i.e., away from the void 1 1) flexation of the cover 12 induced by fluid pressure within the apparatus.
  • Supports 20 can have rectangular, rhomboid, circular, elliptical, or wing-shaped cross- sections, for example.
  • supports 20 can induce turbulence in fluid flow paths and induce mixing and or displacement of particles immediately downstream from such supports.
  • supports having rounded cross-sections and placed near the leading (i.e., upstream-most) edge of the second passage 52 can induce turbulent flow at the leading edge of the second passage 52, jostling particles that might otherwise occlude the second passage 52 and thereby enhancing fluid flow through the second passage 52.
  • the separation element 14 can define fluid flow paths other than the stepped passageway discussed herein. Such fluid flow paths can, for example, extend between the inlet region 15 and the stepped passageway or between the stepped passageway and the outlet region 17. Further by way of example, the first passage 51 defined by the first step 61 of the separation element 14 can be connected with the second passage 52 defined by the second step 62 of the separation element 14 by way of a fluid flow path defined by the separation element (i.e., rather than the first passage 51 communicating directly with the second passage 52).
  • the separation element 14 can define walls or channels that originate at the inlet port 16 and extend by various paths to each of the individual stepped passageways, such that the linear flow distance along each flow path is equal.
  • the flow path extending between the inlet port 16 and the central flow path will be curved, angled, or serpentine relative to the flow paths extending between the inlet port 16 and the outermost flow paths.
  • the separation element 14 includes at least two steps, including a first step 61 nearer (along the stepped passageway) the inlet region 15 than a second step 62. Particles suspended in a fluid flow through the stepped passageway that includes a first passage 51 and a second passage 52 that has a smaller narrow dimension than the first passage 51. Most or all particles in the fluid are able to flow into the first passage 51, but only some of the particles are able to flow through the second passage 52. The net result is that some particles in the fluid can flow through the entire stepped passageway, while other particles are retained within the apparatus, such as within the first passage 51. Segregation of particles is thus achieved.
  • the steps of the separation element 14 can have any of a variety of shapes.
  • the first step 61 and the second step 62 have a traditional 'staircase' step structure, i.e., two planar surfaces that intersect at a right angle. That is, the transitional face 41 of the first step 61 and the broad face 31 of the first step 61 meet at a right angle, as do the transitional face 42 of the second step 62 and the broad face 32 of the second step 62.
  • the transitional and broad faces of the steps can meet at an angle between 90 and 180 degrees, as depicted in Figure 3, for example.
  • the transitional and broad faces of the steps can also meet at an angle between 0 and 90 degrees, forming an overhang.
  • Steps that form an overhang and steps that have faces that meet at angles near 90 degrees can induce turbulent flow near the edge at which the faces of the step meet.
  • Such turbulence can dislodge particles that might otherwise occlude the passage between the broad face of the step and the opposed face of the body 10 or cover 12, and this turbulence can thereby inhibit clogging of the passage and enhance fluid flow (and reduce fluid pressure drop) through the device, which are beneficial effects.
  • steps can also reduce clogging of the passage and improve performance of the apparatus.
  • one or more steps designed to capture or exclude such particles can be incorporated into the device in order to capture the undesired particles in a place and quantity that does not significantly inhibit fluid flow through the stepped passageway.
  • Steps having transitional and broad faces that meet at an angle between 90 and 180 degrees can occlude passage of particles having a variety of sizes (i.e., those having sizes intermediate between the narrow dimension of the passage defined by the broad face of the step and the narrow dimension of the space upstream from the step.
  • a step having transitional and broad faces that meet at an angle between 90 and 180 degrees can prevent clogging of the passage defined by the broad face of the step to a greater degree than a step having transitional and broad faces that meet at an angle of 90 degrees or less.
  • Clogging of fluid flow past a step by particles that occlude the passage defined by the broad face of the step can also be reduced or avoided by increasing the width of the step. Because each particle occludes fluid flow only for the flow area obscured by the particle, a wider step will necessarily be clogged by a greater number of occluding particles.
  • the width of a step can be increased in either or both of two ways. First, the width of the step can be increased by simply increasing the linear width (as depicted in Figure 4) of the step. Second, the width of the step can be increased by increasing the length of the edge at which the broad and transitional faces of the step meet by decreasing the linearity (i.e., straightness) of the step.
  • a step that extends directly across (i.e., at right angles to the sides) of the channel has an upstream- most edge with an edge length simply equal to the width of the channel.
  • the shape of the step is a semicircle, with the arc of the semicircle extending such that the center of the semicircle lies downstream from the upstream-most edge of the semicircle, the edge length of the step is equal to the length of the semicircle, which is the number pi multiplied by the width of the channel and divided by two (i.e., roughly 1.57 x the width of the channel).
  • steps having edges shaped like an arc of a circle or ellipse, like chevrons (i.e., like the letter V), like zig-zags, like serpentine lines, or like irregular lines will all have edge lengths greater than the edge length of a step that extends perpendicularly across a fluid channel having a rectangular cross-section. Steps having edges with such shapes can be used in the apparatus described herein.
  • the dimensions of the first step 61 and the second step 62 are not critical, except that the second step 62 defines a boundary of the second passage 52, which serves to segregate particles as described herein. For that reason, the dimensions of the second step 62 and the corresponding second passage 52 defined by the second step 62 of the separation element 14 and the opposed surface(s) of the body 10 or cover 12 should be carefully selected. Criteria relevant to selecting these dimensions include the dimensions of the particles to be segregated by their ability to traverse the second passage 52.
  • the narrow dimension of the second passage 52 should be selected such that the relatively large cells are substantially unable to enter the second passage 52 and that other cells in the population are able to enter and traverse the second passage 52.
  • the shape and width of the second step 62 should be selected based on the number of relatively large cells that are anticipated to be present in the sample, so that clogging of the second passage 52 by the relatively large cells can be reduced, delayed, or avoided.
  • the narrow dimension of the second passage 52 should be selected to closely match the size of the two types of particles, it being understood that although both types of particles will be able to enter the second passage 52, the relatively deformable particles will, on average, be able to traverse the second passage 52 in less time than the particles of limited fluidity.
  • each second passage 52 can also be advantageous for each second passage 52 to have a relatively short length, so as to minimize clogging by the relatively deformable particles, which will traverse the second passages 52 in less time than the particles of limited fluidity.
  • the width (i.e., as defined herein and shown in Figure 4) of the each of the first step 61 and the second step 62 can be selected based on the anticipated accumulation of particles on the step, in view of the sample anticipated to be processed using the apparatus. Based on the narrow dimension of the second passage 52, the proportion and number of particles that will be unable to enter the second passage 52 can be estimated.
  • the width of each step is preferably selected to prevent total occlusion of flow past the step.
  • the width of a step (and the corresponding passage defined by the step) can be selected to be significantly (e.g., 10, 1000, or 100000 times) greater than the narrow dimension of the passage.
  • a step width approximately at least 1000 (one thousand), and preferably 10000 (ten thousand), times the narrow dimension of the corresponding passage is considered desirable. Relatively wide steps permit accumulation of particles within a passage while limiting clogging of the passage.
  • the width and length of the first step 61 can be selected to accommodate the anticipated number of such cells.
  • the length (i.e., as defined herein and shown in Figure 4) of the first and second steps of the separation element 14 are generally not critical, as it is the narrow dimension of the first and second passages (which are bounded by the first and second steps, respectively) that provide the segregative functionality of the apparatus described herein. In situations in which it is desired to accumulate or observe particles on a step, the length of the step can be selected to accommodate the anticipated or estimated number and size of the particles on the step.
  • the length of the step can influence the degree of segregation achieved, longer steps enhancing the segregation effected by differing rates of traversal.
  • Step length can be increased by increasing the length of a single step, by increasing the number of steps of a selected length (each step defining a passage having the same narrow dimension), or by a combination of these.
  • planar step regions can be substantially parallel to a portion of the cover, a portion of the body, or both, and should have a length (in the direction of bulk fluid flow) equal to a multiple (e.g., 2, 4, 10, or 1000) of the narrow dimension of the passage it bounds.
  • the width of the planar region (in the direction perpendicular to bulk fluid flow) should be equal to a multiple (e.g., 10, 1000, of 10000) of the narrow dimension of the passage it bounds.
  • the ratio of the width of the planar region (in the direction of flow perpendicular to bulk fluid flow) ranges from 1,318 at the most open end to 805 at the narrowest (outlet) end; 659 at the most open end to 967 at the narrowest (outlet) end, 537 at the most open end to 725 at the narrowest (outlet) end for each of three separate cassette designs. Gradations on each of the chips increases the ratio of step width to height by 66.7 going from the inlet to the outlet side of the cassette. This width to height ratio will vary depending upon the ratio of the number of particles it is desired to capture within the cassette to those which it is desired to pass through the cassette.
  • the ratio of fetal cells to (white blood cells + red blood cells) that are captured by devices of the type described herein can be quite high, and selection of appropriate step height and length can permit passage of greater than 99.99% passage of all nucleated blood cells in a maternal blood sample.
  • each step defining a passage within the stepped passageway having a characteristic narrow dimension.
  • the apparatus can include a single separation element 14 or a plurality of separation elements 14.
  • the apparatus can include a first separation element that defines a first step 61 and a second separation element that defines a second step 62. If integral with the body 10, the first and second separation elements 14 can be disposed at different locations on the body 10, so long as both separation elements 14 are within the void 1 1, interposed between the inlet region 15 and the outlet region 17 of the void 1 1, and define steps in the same stepped passageway.
  • a separation element defining the first step 61 can be integral (or attached to) with the body 10
  • a second separation element defining the second step 62 can be integral with (or attached to) the cover 12, so long as both separation elements are within the void 11, interposed between the inlet region 15 and the outlet region 17 of the void 1 1, and define steps in the same stepped passageway.
  • the two separation elements can be discrete pieces, provided the same conditions are satisfied.
  • the separation element 14 can be constructed from a unitary piece of material (and can be integral with one of the body 10 and cover 12) or it can be constructed from a plurality of pieces of material.
  • the separation element 14 of an apparatus like the one depicted in Figure 1 can be formed of two rectangular bars (solid forms having three pairs of parallel faces, each pair being oriented at right angles to the other two pairs) of material, one bar lying atop a flat portion of the body 10 in the void 11 and forming the first step 61, and the second bar lying atop the first bar and forming the second step 62.
  • each step should be selected such that at least some particles will be able to pass through the passage defined by that step, and at least some other particles will not be able to pass through the passage defined by that step.
  • a rigid particle's ability to pass through a passage depends on the characteristic dimensions of the particle.
  • a rigid particle cannot pass through a passage that has a height which is less than the short dimension of the particle.
  • a rigid particle will be substantially uninhibited from passing through a passage that has a height which is greater than the long dimension of the particle.
  • a rigid particle can pass through a passage that has a height which is greater than its short dimension but less than its long dimension, but the passage will at least somewhat inhibit the particle from passing.
  • deformable particles e.g., biological cells, gas bubbles, or cereal grains
  • deformable particles can traverse passages having narrow dimensions smaller than the short dimension of the particle, to the extent the particle can deform to 'squeeze' through the passage. This ability depends on the rigidity of the particle, the size of the passage, and the fluid pressure applied against the particle. Where these quantities are not known or predictable, empirical data can be gathered to determine or estimate the ability of such particles to traverse a passage of a given size, and such empirical data can be used to select appropriate dimensions for the first and second passages of the apparatus described herein.
  • fluid passages having rectangular cross-sections such cross sections taken perpendicular to the direction of bulk fluid flow.
  • the fluid passages of the apparatus described herein are not limited to such rectangular channels.
  • the walls of the fluid passages can be perpendicular to one another and to one or more of the body 10, cover 12, and separation element 14.
  • the walls can have other arrangements as well.
  • the fluid passages are rounded, such as passages formed by removal of material by a spinning bit having a rounded tip.
  • fluid passages can be rounded on one side (e.g., where formed into the body 10) and flat on another side (e.g., where bounded by a flat cover 12).
  • Fluid shear stresses can harm deformable or breakable particles, such as biological cells. Reduction of fluid shear stresses within the apparatus is therefore desirable when the apparatus is to be used to process such particles.
  • Significant fluid shear stress can occur at positions in fluid channels at which the linear flow velocity changes rapidly, such as at locations at which the geometry of the fluid channel changes.
  • the geometry of the fluid channels can be selected to increase, decrease, or maintain constant the linear flow velocity within the apparatus.
  • Increasing or decreasing linear flow velocity creates fluid shear stress.
  • the level of fluid shear stress can be selected to rupture, deform, or destroy some kinds of particles over other kinds of particles. For example, durable particles can be segregated from breakable particles having the same size by inducing fluid shear stress that ruptures the breakable particles.
  • the durable particles are retained in the passageway while the fragments of the breakable particles pass the second step 62 and flow into the outlet region 17.
  • substantially constant linear fluid velocity can be maintained throughout the apparatus (or at least throughout the stepped passageway thereof) by selection of appropriate fluid channel dimensions.
  • the body 10, cover 12, and separation element 14 can be formed such that the cross-sectional area of the stepped passageway with respect to the direction of fluid flow increases, decreases, or remains constant.
  • the cross-sectional area of the stepped passageway affects the pressure and flow rate of the fluid in the apparatus. If the separation element has a constant width, then the cross-sectional area defined by the height and width of the first passage 51 will be smaller than the cross-sectional area of the inlet region 15.
  • the cross-sectional area of the second passage 52 (e.g., defined by the height and width of the second passage if it is rectangular in cross section) will be smaller than that of the first passage 51.
  • the geometry of the fluid channels can be selected to counteract these changes in fluid pressure and flow rate.
  • the width of a passage having a rectangular cross section can increase proportionally as the height of the passage decreases, such that the cross-sectional area of passage is constant.
  • the width of the passage defined by the transition face can increase at a constant rate, equal to the rate at which the height of the passage decreases.
  • the fluid pressure and flow rate through the passageway defined by such a separation element remains constant.
  • An example of such a passageway is shown in Figure 3.
  • the body 10, cover 12, and separation element 14 can be formed such that fluid flux is equal at all places throughout the narrow passageway of the apparatus.
  • fluid flux throughout the inlet region 15, the passages defined by surfaces 41, 31, 42, and 32, and the outlet region 17 can be constant.
  • the body 10, cover 12, and separation element 14 can be formed such that fluid flux increases or decreases in the direction of bulk fluid flow.
  • the surfaces of the body 10 or cover 12 that define the width of the void 11 can taper in the direction of the inlet region 15 or outlet region 17.
  • Fluid shear stresses are, of course, not a concern when the apparatus is operated without a fluid in the stepped passageway. Because the viscosities of gaseous fluids are substantially lower than the viscosities of liquid fluids, fluid shear stresses are of lesser concern when the particles are suspended in a gaseous fluid (e.g., air) than in a liquid fluid. Similarly, because fluid shear stresses vary in known ways with fluid viscosity, modifications of the apparatus described herein suitable for accommodating fluids of different viscosities will be apparent to the ordinarily skilled designer.
  • the body, the cover, or both can have one or more fluid channels that fluidly connect with the surface of a step of the separation element, for removing fluid from the step (including any cells suspended in the fluid upon that step). Furthermore, when the step has regions or discrete grooves in the step, the cover or body can be machined so that the fluid channels fluidly communicate most nearly with a discrete groove or region upon the step, for removing fluid in the vicinity of that groove or region of the step. Such local channels can improve purification by capturing only a relatively small amount of fluid in the immediate vicinity of the channel when a particle is captured thereby.
  • the body, the cover, the separation element, or some combination of these can have an optical, electrical, or optico-electrical device constructed therein or thereon (e.g., by etching, film deposition, or other known techniques) at a position that corresponds to a selected step or a selected groove or region of a step.
  • Such devices can be used to detect cells (e.g., using a detector to detect a decrease in light or other radiation transmitted across the fluid between the surface of the step and the cover or body) or to manipulate cells (e.g., using an activatable heating element to ablate cells which pass or rest near the heating element).
  • Devices constructed upon the cover, the body, or the steps can be made individually activatable by assigning an electronic address to the device. In this manner, cells can be detected at discrete areas of the device, and cells at selected areas can be manipulated without manipulating cells at other positions.
  • Harvesting of cells from a selected step can be performed by simply withdrawing fluid from that step or a portion of the step.
  • energy can be applied in many forms, and a preferable form will usually depend on the type of cell or object to be displaced and the identity of the force or phenomenon which inhibits removal of the cell or object from the step.
  • withdrawal of fluid from one portion of a step can be performed simultaneously with addition of fluid at another portion of the same step.
  • forms in which energy can be applied to the apparatus in order to harvest cells include shaking, tapping, or vibrating the apparatus, or applying energy in the form of ultrasound, heat, infrared or other radiation, bubbles, compressed air, and the like.
  • the cells can instead be detected or manipulated.
  • one or more cells are lysed by application to the cells of electrical, mechanical, or heat energy, thereby releasing the contents of the cell in the void of the apparatus.
  • the cell contents can be analyzed or manipulated in the apparatus, or they can be recovered from the apparatus and analyzed or manipulated outside of the apparatus.
  • a cell that is retained at a particular location on a step can be lysed using a device located at or focused upon that particular location, thereby releasing the cell's DNA into the void.
  • the DNA can be amplified in the void by providing PCR reagents to the void, or it can be collected (e.g., in a container in which fluid obtained from a selected portion of the void is collected or, alternatively, by passing fluid through the void and collecting the DNA in the outlet fluid) and amplified outside of the apparatus.
  • the apparatus can thus be used to analyze the contents of individual cells or groups of cells.
  • any of a wide variety of methods for harvesting or manipulating cells within a device can be employed using the apparatus described herein.
  • methods employing known "optical tweezer" devices, laser microdissection devices, and particle- binding membranes and films can be employed.
  • the film or membrane can overlie an orifice or fluid channel, sealing the orifice or fluid channel from the remainder of the void.
  • the portion of the film or membrane contacting the particle can be detached or punctured, placing the particle in fluid communication with the orifice or fluid channel previously segregated by the film or membrane.
  • the detached portion of the film or membrane e.g., having a particle of interest attached thereto
  • the detached portion of the film or membrane can be isolated either by screening for a characteristic of the particle or for a characteristic (e.g., a spectrophotometric property or magnetic property) of the film or membrane.
  • a property e.g., magnetism
  • the film can be used to mechanically manipulate particles attached to it.
  • a detached portion of a magnetic film or membrane having a cell attached to it can be used as a transportation vehicle for that cell by applying a directional magnetic field to a fluid in which the membrane is suspended or by moving a magnetic probe to guide the detached portion of the magnetic film or membrane with cell attached to it towards a desired location such as a channel, chamber or container.
  • material(s) used to construct the body 10 and the cover 12 are not critical, except that they should be sufficiently rigid that the parts will maintain their shapes, and not substantially deform or break, during operation of the apparatus as described herein. Where deformable materials are used, the expected deformation under conditions of operation should be taken into account when designing the size and shapes of the parts.
  • suitable materials include glasses, solid polymers such as
  • the body 10, cover 12, separation element 14, and other components described herein can each be formed from a different material, if desired.
  • all parts are formed of the same material, so that the effects of, for example, temperature, on expansion and contraction of parts is similar for all parts.
  • At least one of the body 10 and the cover 12 should be constructed from a material that facilitates observation of the particles in the assembled apparatus.
  • many glasses are transparent to wavelengths of light in the region of the optical spectrum that is visible to the human eye. Construction of one or more parts of the apparatus from such a glass permits an operator to visually inspect particles in the void (e.g., accumulation of particles in the first passage 51) during operation of the apparatus.
  • the identity of the materials used to construct the separation element 14 is also not critical, except that it should be sufficiently rigid that the separation element 14 will maintain its shape, and not substantially deform or break, during operation of the apparatus as described herein.
  • Selection of materials used to construct the apparatus and its parts can be influenced by the nature of the particles to be segregated therein.
  • the nature of the particles can also influence decisions regarding which, if any, surface treatments may be appropriate for modulating interaction of particles with surfaces they may encounter within the device. For example, if particles are to be segregated within the device without substantially binding or adhering to the device, then the materials and/or surface treatments should be selected to reduce or eliminate the likelihood of particle binding to the surfaces.
  • one or more surfaces of the device e.g., the broad surface 31 of the first step 61
  • biological cells are known to express a variety of proteins on their surface, and antibodies that specifically bind to a protein of a selected type can be generated by known methods. If antibodies that specifically bind to a protein expressed on the surface of cells of a particular type are fixed to a surface in the stepped passageway, binding of the cells of the particular type with the antibodies can be expected to inhibit or halt passage of the cells past the surface in the apparatus, enhancing the segregation of those cells from cells that do not express the protein on their surface (and to which the antibodies cannot bind).
  • Selection of methods to construct the apparatus can be influenced by the size of the particles to be separated therein.
  • the particular method employed to construct the apparatus and its parts is not critical.
  • a wide variety of methods of forming parts having shapes and conformations that are accurate to the micrometer and nanometer scale are known.
  • any of a variety of known micromachining methods can be used. Examples of such micromachining methods include film deposition processes, such as spin coating and chemical vapor deposition, laser fabrication, and photolithographic techniques such as UV or x-ray processes, precision machining methods, or etching methods which may be performed by either wet chemical processes or plasma processes. (See, e.g., Manz et al, 1991, Trends in Analytical Chemistry, 10: 144-149).
  • the parts can be molded, rather than machined, using any of a variety of known molding methods.
  • a wide variety of methods of forming and machining parts for use on a macroscopic scale are known, such as cutting, carving, molding, engraving, welding, and casting.
  • the body 10, cover 12, and separation element 14 can be constructed separately and assembled to form the apparatus, and such assembly can be performed by the manufacturer or the user of the apparatus.
  • the separation element 14 can be constructed as an integral part of one of the cover 12 or the body 10.
  • a single cover 12 is made capable of sealing a void 11 formed with any of a variety of bodies 10 (e.g., each having a separation element 14 in the void 11 of the body 10, the various separation elements 14 having different properties, such as different step heights).
  • the apparatus segregates particles based on the ability of various particles to traverse the first and second passages of the apparatus described herein.
  • the particles that can be segregated using the apparatus include living particles such as animal or plant cells, bacteria, or protozoa, or non-living particles.
  • the apparatus described herein can be used to segregate larger particles (e.g., cereal grains, rodent feces, gas bubbles, and bowling balls) and smaller particles (e.g., subcellular organelles, viruses, and precipitated mineral particles).
  • Attributes of the particles that affect their ability to traverse the first and second passages of the apparatus described herein include the size, shape, surface properties, and deformability of the particles.
  • a particle tumbling randomly in a fluid will sweep out an exclusion volume equal to the volume of a sphere having a diameter equal to the longest dimension of the particle.
  • a rigid sphere having a diameter of 1 micrometer, a randomly-tumbling disk- shaped rigid particle having a diameter of 1 micrometer and a thickness of 0.2 micrometers, and a randomly-tumbling rod-shaped rigid particle having a length of 1 micrometer and a diameter of 0.1 micrometer will each sweep out an equal exclusion volume. Ignoring the effects of surface properties, each of these particles will be able to traverse a passage having a narrow diameter greater than 1 micrometer.
  • the disk-shaped and rod-shaped particles will be able to traverse a passage having a narrow diameter less than 1 micrometer and greater than 0.2 micrometer.
  • the rod-shaped particles will be able to traverse a passage having a narrow diameter less than 0.2 micrometer and greater than 0.1 micrometer.
  • the ability of non-rigid (i.e., deformable) analogs of these particles to traverse one of these passages (and the rate at which such traversal can occur) depends on the degree and extent of
  • the surface properties of the particles and the surfaces that define the passage can affect the rate at which the particles traverse the passage, and can prevent such traversal from occurring (e.g., if the particle binds avidly with the surface of the passage or if the surfaces of the passage and the particle repel one another).
  • the particles that are separated are biological cells present in a mixed population of cells (i.e., a suspension of cells that include cells of multiple types). Selection of appropriate narrow dimensions for the first and second passages of the apparatus described herein permits segregation of biological cells based on their size, shape, surface properties, deformability, or some combination of these properties.
  • biological cells that can be separated using the apparatus described herein include fetal cells circulating in maternal blood, embryonic stem cells (in maternal blood or an individual's own embryonic stem cells), adult stem cells, tumor cells, bacteria and other pathogens, and cells of the immune system (e.g., various white blood cells such as T cells, B cells, neutrophils, macrophages, and monocytes).
  • the methods can be used to segregate mixtures of cells of these types.
  • the methods described herein can be used to segregate subcellular organelles (e.g., nuclei, chloroplasts, and mitochondria) as well.
  • the apparatus is used to isolate agents of infectious diseases (e.g., bacteria or viruses) or other pathogens (e.g., protozoa or parasites) from a sample.
  • infectious diseases e.g., bacteria or viruses
  • pathogens e.g., protozoa or parasites
  • the apparatus can be used for diagnostic purposes, such as analyzing a biological sample obtained from a subject in order to determine whether the subject is infected with an infectious agent.
  • a sample such as a water sample or a food product or ingredient can be assessed by using an apparatus described herein to assess the sample directly, or a fluid with which the sample is contacted, for the presence of a pathogen which, if ingested by a subject, would contribute to the likelihood that the subject would develop a disease or other condition.
  • stem cells can be segregated from other cells present in maternal blood or in placental blood.
  • blood includes a variety of cells, including stem cells, red blood cells, and platelets.
  • blood is preferably collected upstream of capillary beds when the cells that are sought have a size (i.e., diameter > 8 - 10 micrometers) exceeding the normal diameter of capillaries.
  • arterial blood e.g., blood taken from the common hepatic artery
  • a fluid derived from such pre-capillary blood e.g., lung and bronchial exudates and secretions, or fluids containing them, such as bronchial lavage fluids
  • large cells such as fetal trophoblasts and stem cells.
  • Human stem cells tend to exhibit an exclusion volume equal to a sphere having a diameter of about 12 micrometers.
  • Human red blood cells tend to exhibit an exclusion volume equal to a sphere having a diameter of about 5.5 micrometers.
  • Human platelets tend to exhibit an exclusion volume equal to a sphere having a diameter of about 1 micrometer.
  • the stem cells, but not the red blood cells or platelets will be excluded from a passage having a narrow dimension on the order of 4 to 8 micrometers.
  • Stem cells provided to the inlet region 15 of an apparatus described herein with a second passage 52 having a narrow dimension of 4 to 8 micrometers will generally not pass to the outlet region 17 of the apparatus, although red blood cells and platelets will.
  • the narrow dimension of the first passage 51 is greater than about 12 micrometers (e.g., if the narrow dimension of the first passage 51 is 18 micrometers), then stem cells, red blood cells, and platelets will all traverse the first passage 51.
  • red blood cells and platelets will pass through (i.e., through the first and second passages to the outlet region 17 of) the apparatus, while stem cells will be retained upstream from the second passage 52.
  • an apparatus configured such as the one depicted in Figure 1 i.e., wherein there is no intervening passage or chamber between the first and second passages)
  • the stem cells will accumulate in the first passage 51.
  • Particles within the stepped passageway are subjected to shear, compressive, and other forces acting upon them by any fluid flowing through the passageway. If particles (e.g., biological cells) that exhibit different resistant to deformation, compression, bursting, lysis, or breakage (i.e., any characteristic that alters the rate or ability of the particle to traverse one or both of the first and second passages) are present, the differences in response of the particles to fluid flow can be used to differentially affect passage (or non-passage) of the particles through the stepped passageway.
  • particles e.g., biological cells
  • breakage i.e., any characteristic that alters the rate or ability of the particle to traverse one or both of the first and second passages
  • these two types of cells can be separated from other particles under conditions of relatively low fluid flow (i.e., flow low enough that few or no cells lyse). After such separation, the fluid flow rate can be increased in order to generate sufficient fluid shear within at least one portion of the stepped passageway that cells of the first type, but not cells of the second type, will lyse, yielding first cell type lysis products in the effluent from the outlet region and cells of the second type retained within the apparatus.
  • the apparatus described herein can be operated by providing particles to the inlet region 15 of the void 11 of the apparatus and permitting the particles to move through fluid present in the inlet region 15, the stepped passageway, and the outlet region 17, such movement being attributable to intrinsic motility of the cells or to passive settling of non- motile particles under the influence of gravity.
  • the apparatus will need to be oriented such that gravity will tend to cause particles that are denser than the fluid to 'fall' from the inlet region 15, through the stepped passageway, and toward the outlet region 17 or, for particles that are less dense than the fluid, to cause the particles to 'rise' from the inlet region 15, through the stepped passageway, toward the outlet region 17.
  • the apparatus described herein is operated by fluidly connecting a reservoir containing a fluid (e.g., a particle-containing suspension or a particle-free fluid) or another fluid displacement device such as a pump to the inlet region 15.
  • Fluid flow through the apparatus is achieved by introducing fluid at the inlet region 15 of the apparatus, by continuously withdrawing fluid from the outlet region 17 of the apparatus, or both. Fluid introduced at the inlet region 15 displaces fluid already present within the void 1 1 and induces emission of fluid from within the void 1 1 into the outlet region 17 or through an outlet port 18 that fluidly communicates with the outlet region 17. As particles traverse the stepped passageway of the apparatus, they will emerge therefrom into the outlet region 17.
  • Such particles can be recovered from fluid that accumulates within the outlet region 17 or a reservoir that fluidly communicates with it or from fluid that is withdrawn from an outlet port 18 that fluidly communicates with the outlet region 17. Particles that are unable to traverse either the first passage 51 or the second passage 52 of the apparatus during fluid flow through the apparatus will be retained within the apparatus and can be recovered therefrom.
  • the identity of the fluid displacement device that is used to provide fluid flow to the inlet region 15 is not critical.
  • the fluid displacement device can be simply a reservoir containing fluid that is permitted to drain, under the influence of gravity, through the apparatus by way of a fluid connection between the reservoir and an inlet port 16 that fluidly communicates with the inlet region.
  • a mechanical pump can deliver fluid to the inlet port 16 by way of a sealed fluid connection between the pump outlet and the inlet port 16. Fluid delivered by the pump displaces fluid present in the inlet region 15 of the apparatus into the stepped passageway and thence toward the outlet region 17, from which displaced fluid can be withdrawn, collected, or emitted.
  • a mechanical pump can withdraw fluid, by way of a sealed fluid connection, from an outlet port 18 in fluid communication with the outlet region 17 of the apparatus. Withdrawal of fluid from the outlet region 17 lowers the fluid pressure at the outlet region 17, inducing displacement of fluid from the adjoining stepped passageway of the apparatus into the outlet region 17 and from the inlet region 15 into the stepped passageway.
  • Positive displacement of fluid in the void 11 of the apparatus increases fluid pressure within the void.
  • Increased fluid pressure can alter the dimensions of the apparatus (e.g., by inducing flexion or displacement of parts of the apparatus), the dimension of particles within the apparatus (e.g., deformable gas-filled particles will tend to decrease in size as the surrounding fluid pressure increases), or both.
  • pulsating or otherwise varying fluid pressure can induce transient changes in localized fluid flow within the apparatus.
  • Transient localized flow variations can be beneficial. For example, particles which are unable to enter the first or second passage of the stepped passageway can be urged against the upstream extent of the passage, blocking fluid flow through the portion of the passage occluded by the particle. Transient variations in flow of fluid at the point of occlusion of the passage by the particle can alternately urge the particle against the passage opening and urge the particle away from the opening, thereby temporarily relieving the occlusion and permitting fluid flow through the previously-occluded portion of the passage.
  • Fluid pulsations or other rapid flow changes can induce shear stresses in the fluid and upon particles suspended in the fluid, and particles can be damaged by such shear stresses.
  • Particle damage e.g., lysis of biological cells
  • alterations in the types and characteristics of fluid displacement devices connected with the apparatus can increase or reduce shear stresses within the fluid.
  • pumps which deliver fluid at a relatively constant volumetric rate i.e., rather than a more pulsatile volumetric rate, as with many peristaltic pumps
  • pumps which deliver fluid at a relatively constant pressure can reduce fluid shear stresses that would otherwise build as portions of the first and/or second passage of the stepped passageway become occluded with particles or debris if volumetric flow rate were not adjusted accordingly.
  • An example of a pump suitable for moving fluid through the apparatus is a low pulse syringe pump.
  • Such a pump can include an agitation mechanism, which may be useful to prevent particles from settling during operation of the apparatus.
  • Negative displacement of fluid from within the void 1 1 reduces fluid pressure within the void 1 1 and can induce similar difficulties, including deformation and displacement of parts of the apparatus and transient flow variations. Negative displacement of fluid from the void 1 1 can also induce bubble formation within fluid in the apparatus, and bubbles can disrupt operation of the apparatus (e.g., by occluding fluid flow through a portion of a passage or by inducing surface tension-related effects upon particles in the apparatus). Bubble formation should therefore be avoided. Positive fluid displacement of fluid within the void 1 1 of the apparatus is preferred for this reason.
  • fluid is displaced through the apparatus by application of centrifugal "force” to a fluid-containing reservoir in fluid communication with the inlet region 15 of the apparatus.
  • Centrifugal "force” is generated by spinning the reservoir about an axis, and conservation of angular momentum of the fluid urges the fluid away from the axis of rotation.
  • This "force” can be used to displace fluid from the void 1 1 of the apparatus by fluidly connecting the reservoir outlet with the inlet region 15 of the apparatus.
  • an centrifugally-operable apparatus can include, in a linear arrangement from a position proximal to the axis of rotation toward a position distal to the axis of rotation, a fluid reservoir, the inlet region 15 of the void 1 1, the stepped passageway, and the outlet region 17 of the void 1 1.
  • Fluid from the reservoir is driven by centrifugal "force" into the inlet region 15, thence through the stepped passageway (the first passage 51 being located proximal to the axis of rotation relative to the second passage 52), and thence to outlet region 17, which can include a second reservoir for collecting fluid that has passed through the apparatus. Particles unable to traverse the second passage 52 will remain within the void 1 1 after some or all of the fluid in the fluid reservoir has passed through the apparatus.
  • the apparatus includes at least a cover 12 and a body 10 that are assembled to yield an operable device and because, in operation, positive internal fluid pressure is exerted within the apparatus that would tend to separate the cover 12 and body 10, some means of clamping or otherwise holding the body 10 and cover 12 in their assembled position is usually employed. Pressures induced by clamping or otherwise holding the cover 12 and body 10 in their assembled positions can induce deformation of the parts of the cover 12 or the body 10, potentially altering the significant dimensions of the parts. It is important to detect such deformation when it occurs.
  • the disclosure includes a method of confirming appropriate assembly of the apparatus described herein.
  • This method is exemplified for an apparatus that includes a body 10 that defines a void 1 1 and a cover 12 that covers the void 1 1 and has a flat surface opposite the face that covers the void 11.
  • substantially the same method can be used to detect deformation in a part for other configurations by including a flat surface on the face of a part in which deformation is to be detected.
  • the body 10 and cover 12 are assembled, including all clamps, holders, or other devices that exert pressure upon any portion of the body 10 or cover 12.
  • a particle- free fluid is flowed through the apparatus at the operating pressure to be used.
  • the flat surface of the cover 12 is illuminated with radiation.
  • the interference pattern of radiation reflected or refracted by the flat surface of the cover 12 is examined.
  • the interference pattern indicates the location and extent of bending in the cover and permits confirmation, for example, of whether the variation in the distances between the face of the cover 12 that defines the void 1 1 and the walls of the void 11 defined by the body 10 is within the appropriate tolerance.
  • the apparatus can include a variety of visual indicators that confirm proper assembly of the apparatus.
  • a visual indicator is a feature of the body or cover that has one appearance when the apparatus is properly assembled, and a different appearance when the apparatus is not properly assembled. Substantially any visually-observable phenomenon can serve as the visual indicator. As indicated above, interference patterns indicating deformation of a part of the apparatus can be used. Alignment of lines drawn, painted, or inscribed on mating parts can serve as a visual indicator of proper assembly.
  • the apparatus can be used to segregate particles, such as biological cells, that are suspended in a fluid sample.
  • the fluid sample is introduced at the inlet region 15 of the void 1 1.
  • Particles in the sample move from the inlet region 15 into a stepped passageway defined by the separation element 14 and at least one of the body 10 and the cover 12. Movement of the particles within the apparatus occurs by virtue of inherent motility of the particles (e.g., for motile biological cells), by density-mediated settling or rising of particles through the fluid within the apparatus, or in response to bulk fluid flow that is induced within the apparatus.
  • the stepped passageway includes a first passage 51 that is bounded by a first step 61 of the separation element 14.
  • the first passage 51 has a narrow dimension (i.e., the distance between the surface of the first step 61 and the opposed face of the body 10 and/or cover 12), and some particles may be unable to enter the first passage 51 on account of their size (taking into account the deformability of the particle). Particles that are able to traverse the first passage 51 continue to move along the stepped passageway to a second passage 52 that is bounded by a second step 62 of the separation element 14.
  • the second passage 52 has a narrow dimension (i.e., the distance between the surface of the second step 62 and the opposed face of the body 10 and/or cover 12) that is narrower than the narrow dimension of the first passage 51, and some particles may be unable to enter the second passage 52 on account of their size (taking into account the deformability of the particle). Particles that are able to traverse both the first passage 51 and the second passage 52 continue to move along the stepped passageway to the outlet region 17 of the void 11. The apparatus thus segregates particles unable to enter the first passage 51, particles able to traverse the first passage 51 but unable to enter the second passage 52, and particles able to traverse both the first passage 51 and the second passage 52.
  • populations of particles can be separately recovered, as can particles able to enter, but not traverse (during the period of operation) one of the first and second passages.
  • effluent recovered from the outlet region of the apparatus can be recovered.
  • particles unable to traverse one or both of the first and second passages can be lysed or otherwise degraded (i.e., to permit the lysis or degradation products to pass through the device) prior to recovering the effluent.
  • Multiple apparatuses can be operated at once (i.e., simultaneously), with the same fluid sample applied to the inlet region 15 of each apparatus.
  • the multiple apparatuses can have a common inlet region 15 or a common upstream reservoir that fluidly communicates with each of the inlet regions 15. It is immaterial whether the multiple parallel apparatuses share the same body 10, the same cover 12, or both.
  • a plurality of discrete apparatuses can be operated independently, of course.
  • a plurality of apparatus are grouped, bonded, or pressed together to form a mass (e.g., a block of wafers, each wafer acting as a body 10 for one apparatus on one face of the waver and a cover 12 for an adjacent apparatus on the opposite face of the wafer) having the inlet regions 15 (or fluid channels that fluidly communicate with the inlet regions 15) at one end of the mass.
  • a fluid sample including particles can be applied to the end of the mass, and the fluid sample can thereby be provided to the inlet region 15 of each apparatus of the mass.
  • Fluid flow can be induced through all of the apparatuses of the mass by providing fluid to the same end of the mass under pressure (e.g., using a pump). This arrangement allows scale-up of the apparatus and methods described herein without re-engineering or redesign of the components of the apparatus. Instead, the number of wafers can simply be increased to accommodate the anticipated number of particles.
  • Particles and cells obtained using the apparatus and methods described herein can be used for any of a wide variety of further purposes. Furthermore, for many of those purposes, it is not necessary to isolate particles that may remain within the apparatus after its operation for segregation purposes.
  • reagents e.g., antibodies, enzyme substrates, potentially complementary nucleic acids, and nutrients
  • the fluid channels present within the apparatus can facilitate delivery of such reagents to the cells that remain within the apparatus.
  • the apparatus can be used both to segregate cells and, thereafter, as a reaction vessel to observe interactions of cells with various reagents.
  • the fluids should preferably be selected to have an osmolarity sufficient to maintain the integrity of the biological cells. If viability or other biological functions of the cells are considered important, then the fluids should also be selected so as to maintain the desired biological function(s).
  • the apparatus having particles remaining within it can also be used as a container for storing, maintaining, or contacting reagents with the particles.
  • the apparatus can be used to segregate within the apparatus bacteria that occur in a sample (e.g., a fluid sample with which a foodstuff such as a chicken egg is washed). After segregating the bacteria within the apparatus, growth media can be provided to the void 11 of the apparatus to encourage survival and multiplication of the bacteria.
  • Indicators e.g., antibodies that specifically bind a particular bacterial antigen or a reagent that is metabolizable only by harmful bacteria
  • Such an example is useful for analysis of contamination of the foodstuff with pathogenic bacteria.
  • the apparatus described herein can be operated conveniently by an operator having relatively little expertise, the apparatus can be used to analyze a blood sample at a time very near the time blood is obtained from a subject, such as within a doctor's office or at a phlebotomy laboratory.
  • This disclosure includes descriptions of apparatus and method for segregating from the blood of a woman relatively large cells, such as fetal stem cells and fetal trophoblasts. Blood can potentially contain relatively large cells of non- fetal origin. It can be desirable to assess whether cells isolated from a blood (or other) sample as described herein are of fetal origin.
  • fetal cell markers on cell surfaces can be used to identify a fetal cell.
  • a fetal cell necessarily includes genomic DNA sequences derived from each of the father and the mother of the fetus
  • fetal cells can be differentiated from maternal cells in a sample obtained from the mother of the fetus by detecting the presence of paternal genetic material in a fetal cell.
  • fetal cells derived from a fetus implanted in a woman who is not the genetic mother of the fetus can be differentiated from the woman's cells by detecting the presence of either maternal or paternal genetic material.
  • Substantially any method of detecting paternal DNA or genetic markers in a cell can be used to identify fetal cells in a sample taken from a woman.
  • a fluorescent in situ hybridization method can be used to detect a paternal DNA sequence, such as any sequence on a Y chromosome (which can derive only from the father of a fetus). Any other genetic locus at which the DNA sequence of the paternally-derived allele of a fetus' genome differs from the maternally-derived allele can similarly be used to differentiate fetal cells from maternal cells in a sample.
  • fetal cells can be assessed at multiple loci, especially including loci having high genetic variability within a population that includes the father and mother. If genetic markers other than sequence (e.g., DNA methylation patterns) can differentiate between maternally- and paternally-derived genetic material in a fetal cell, those markers can be used to differentiate maternal and fetal cells in a sample, at least to the extent the genetic markers differ between the father and mother.
  • sequence e.g., DNA methylation patterns
  • Paternal genetic sequences and markers need not be known with certainty in order to differentiate fetal cells and maternal cells in a sample. It can be sufficient to determine that a genetic marker in a cell of interest differ from the corresponding genetic marker in maternal cells.
  • DNA sequences characteristic of Y chromosomes should not occur in any maternal cells (except, of course, in rare cases of XXY trisomy, for example).
  • occurrence of an allele not possessed by maternal genetic material at a locus of a cell of interest identifies the cell as non-maternal.
  • a drawback of identifying a cell simply as not-of-maternal-origin is that the cell may not be a fetal cell but may, for example, be a pathogen or parasite or an artifact of a blood transfusion, animal or human tissue graft, a diseased maternal cell (e.g., one in which a chromosomal translocation has eliminated a maternal allele used as a detection target), or other non- fetal cell.
  • a diseased maternal cell e.g., one in which a chromosomal translocation has eliminated a maternal allele used as a detection target
  • An apparatus of the type disclosed herein was used to separate fetal-like, large nucleated cells from other cells in a 1 milliliter sample of maternal blood.
  • the polycarbonate apparatus was constructed using a known epoxy resin casting process and included a body 10 having an integral separation element 14 in each of eight channels defined by the body 10.
  • Other materials acceptable for this application include cyclic olefin copolymers, and polypropylene cyclo-olefin polymer.
  • the separation element had six steps defining serially-arranged passages in a stepped passageway, the passages having narrow dimensions of 10, 7, 5, 4, 3, and 2 micrometers, respectively. Each step (and passage) had a length of 1 millimeter.
  • a standard glass microscope slide clamped to the body 10 was used as a cover 12. Portions of the body 10 between the discrete stepped passageways served as supports 20.
  • the cover 12 was bonded to the body 10 using silicone rubber adhesive.
  • a sample of blood from a male fetus was obtained and mixed with blood obtained from a woman. This mixture was heparinized using a standard procedure and refrigerated overnight. Other anticoagulants, such as potassium EDTA, are also suitable for this application.
  • the sample was brought to room temperature and injected into the inlet region 15 of a plurality of channels using a syringe. After the sample passed through the apparatus, the apparatus was observed under a microscope. Large cells (i.e., cells larger than normal blood cells) that appeared to be of fetal origin were observed to have been trapped as several positions within the stepped passageways.
  • the large cells were adhered to the glass cover by briefly centrifuging the assembled apparatus. Following centrifugation, the cover 12 was removed from the body 10 and cells adhered to the cover 12 were fixed by Carnoy fixation using a 3 : 1 mixture of methanokacetic acid. The cells were then processed with a standard fluorescence in-situ hybridization (FISH) protocol for detection of chromosomes X and Y using a commercially available kit.
  • FISH fluorescence in-situ hybridization
  • Fluorescent signals representing the hybridization of the FISH probe to site specific sequences on the X and Y chromosomes were observed on the slide, indicating that male (i.e., Y chromosome-containing) fetal cells had been segregated from the blood sample using the apparatus. At least some of the large cells were observed to be polynucleate, suggesting a trophoblastic origin.
  • Fetal trophoblastic cells are believed to be eliminated from maternal blood relatively rapidly following cessation of pregnancy, unlike other types of fetal cells that may occur in maternal blood (e.g., primitive fetal stem cells). Because trophoblastic cells from previous pregnancies are unlikely to persist in the blood of women, segregation of fetal trophoblastic cells can be more informative regarding the status of the woman's current fetus than segregation of other types of fetal cells (including those which may have persisted from previous pregnancies, known or unknown to the woman).
  • Assessing assembly of an apparatus described herein can be achieved by observing light reflected, refracted, or both reflected and refracted from the apparatus under illumination.
  • Figure 5 is a color image which depicts the pattern of light observed on an appropriately assembled apparatus.
  • the apparatus shown in Figure 5 is formed of a plastic body having a separation element integral therewith and having a flat glass cover applied thereto.
  • a stepped passageway is defined by the cover on the (here) upper face of the stepped passageway and by the separation element on the (here) lower face of the stepped passageway.
  • Nine supports extend substantially the entire length of the separation element, from the inlet region (in the direction of the arrow shown in Figure 5) to the outlet region, dividing the stepped passageway into 10 separated flow channels.
  • the separation element has eight flat portions essentially parallel to the cover, the flat portions (steps) defining distances of 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, and 5.4 micrometers from the surface of the cover that defines the stepped passageway.
  • Fluorescent light was emitted from a source at an angle of illumination approximately perpendicular to and directly above the cover.
  • Figure 5 shows the image observed by an observer positioned with a line of sight at approximately 30-45 degrees to the cover. It can be seen that a "checkerboard"-like pattern of light is observed, as shown in Figure 5. Without being bound by any particular theory of operation, it is believed that light reflected from the top (i.e., outside the stepped passageway) surface of the cover combines with light reflected by the bottom surface of the cover, light reflected by the flat portions of the separation element, or some combination of these to yield the colors seen in Figure 5. Regardless of the origin or explanation of the light variations, a pattern of light
  • the apparatus used in the experiments described in this example was a two-piece cassette having a body manufactured from polycarbonate using a micro-injection molding process and a glass cover, the body having a separation thereon defining multiple steps between the separation element and the cover, as shown in Figure 6.
  • the body and cover of the cassette defined a void having an inlet region and an outlet region.
  • the inlet and outlet regions were in fluid communication with each other by way of a separation region.
  • the separation region included a flat segment (i.e., a relatively broad passageway) wherein the minimum distance between the body and the cover was 4.0 micrometers and the maximum distance between the body and the cover was 5.4 micrometers.
  • the cover-to-step distances for the eight steps were (in the direction of fluid flow) 5.4 micrometers, 5.2 micrometers, 5.0 micrometers, 4.8 micrometers, 4.6 micrometers, 4.4 micrometers, 4.2 micrometers, and 4.0 micrometers, as shown in Figure 6.
  • the length of the separation region in the direction of fluid flow i.e., the distance, left-to-right of the 8-stepped structure shown in Figure 6) was 20 millimeters, and each of the eight steps within the separation region had a length, in the direction of fluid flow, of 2.5 millimeters.
  • the width of the separation region (i.e., the distance that the 8-stepped structure shown in Figure 6 extended in the dimension perpendicular to the planar view shown in Figure 6) was 24 millimeters.
  • the total internal volume of the void of the assembled apparatus was about 12.2 microliters, with the volume of the separation region of the void (i.e., the portion between the cover and the stepped separation element) being about 2.2 microliters and the combined volumes of the inlet and outlet regions being about 10 microliters.
  • This model of cassette was designated D3v2.
  • a similar apparatus can be used, the apparatus differing substantially only in that the cover-to-step distances for the eight steps are (in the direction of fluid flow) 4.4 micrometers, 4.2 micrometers, 4.0 micrometers, 3.8
  • the cassette was contained within a purpose- designed holder that served to clamp the cassette and ensure that the glass cover mated with the cassette body in a manner that prevented leakage of any fluid from the cassette.
  • the precise construction of the holder was not critical, and served to apply pressure evenly to the parts of the cassette sufficiently to hold them together and prevent leaks due either to positive or negative fluid pressure within the cassette, relative to atmospheric pressure.
  • the holder was constructed of two metal parts having fittings for adjusting the force with which the metal parts and the cassette parts sandwiched between them were held together.
  • One of the metal parts defined a 'window' (see Figure 5) that approximately corresponded to the void region between the body and cover, through which visual observations of cells within the void could be made.
  • the other metal part was substantially solid, except that it included holes aligned with the inlet and outlet ports to accommodate connections for providing fluid to and withdrawing fluid from the void within the cassette.
  • Fluid flow through the cassette was achieved using a Hamilton PSD3 syringe pump equipped with a 1.25-milliliter syringe.
  • the pump was software-controlled using an application running on MatLabTM Instrument Control Toolbox.
  • the system also includes a pressure sensor that enabled the fluid pressure within the cassette to be constantly monitored.
  • the fluid conduits and fittings with which the components of the system were connected were selected to accommodate anticipated pressures, but their identity was not critical. Substantially any fluid conduits and fittings can be used.
  • the molecular probes used in these studies were obtained from Abbott Molecular and consisted of CEP ® X Spectrum OrangeTM probe (providing a red fluorescence signal from the X-chromosomes in cells treated with the reagent) and CEP ® Y Spectrum GreenTM (providing a green fluorescence signal from the Y chromosome occurring in cells treated with the reagent). All other reagents were of sufficient grade to prevent non-specific hybridization.
  • a CV sample was received in a 15-milliliter screw-cap plastic tube containing pieces of tissue in approximately 5 milliliters of Dulbecco's modified phosphate-buffered saline (DMPBS; 0.90 millimolar CaCl 2 ; 0.49 millimolar MgCl 2 , 2.7 millimolar KC1, 1.47 millimolar KH 2 P0 4 , 138 millimolar NaCl, and 8.06 millimolar a 2 HP0 4 at pH 7.2) in which cells from the tissue sample were suspended.
  • DMPBS Dulbecco's modified phosphate-buffered saline
  • the cell suspension was aspirated, leaving the solid tissue fragments at the bottom of the tube (the volume of material remaining in the tube was less than 0.25 milliliter).
  • the aspirate was placed in a 15- milliliter screw-cap plastic tube and centrifuged at 3,000 rpm (ca. 1,500 x g) for 5 minutes. After centrifugation and removal of the supernatant, approximately 0.1 milliliter of packed cells remained in the tube. Approximately 2 milliliters of DMPBS was added to the tube, and the components were mixed using a vortex-type mixer sufficiently to re-suspend the pelleted cells. This re-suspended cell sample was stored at 4 degrees Celsius for approximately 1 hour.
  • a sample of the re-suspended cell sample was spread on a standard glass microscope slide, stained with Wright-Giemsa stain, and examined at a magnification of 400x under illumination with white light.
  • the stained preparation showed the presence of (fetal) trophoblastic cells in the sample.
  • Other cells that were observed in the sample were believed to be neutrophils (nucleated white blood cells) and red blood cells.
  • Observations of fetal trophoblastic cells and other cells in the sample by this method revealed that the trophoblastic cells were significantly larger than most other cells in the sample.
  • the cassette was dried by applying a vacuum to the outlet region, which effected removal of all fluid from the void in the cassette.
  • the cassette was stored overnight at 4 degrees Celsius.
  • the cassette was microscopically observed at lOOx magnification. Several nucleated cells having a diameter greater than about 20 micrometers were observed in the separation region, some within the inlet region, and others at the first separation step in the separation region of the cassette. No cells having a diameter greater than about 20 ⁇ were observed downstream from the first separation step in the separation region of the cassette.
  • the cassette was disassembled and the glass cover was removed and processed using a standard FISH protocol.
  • the cover was examined using a fluorescence microscope equipped with a computer-controlled stage coupled with an automated detection algorithm.
  • the cover was also stained with DAPI to enable visualization of intact nuclei (i.e., to confirm capture of cells).
  • FISH and DAPI staining were performed as provided in the commercial kit obtained from Abbott Molecular (Chicago, IL).
  • Blood was collected in pairs of approximately 5-milliliter aliquots by venous puncture from each of 22 pregnant women known (by ultrasound imaging) to be carrying a male fetus.
  • the gestational age of the fetuses was within the range from 17 weeks, 6 days and 29 weeks, 6 days, with the average gestational age being 21 weeks, 5 days and the median age being 20 weeks, 2 days.
  • Each blood sample was collected in a 5-millitier tube and was stored in an ice bath until it was prepared for application to the cassette. The time that elapsed between sample collection and sample preparation was less than one hour.
  • cassettes were treated in one of two ways. Some cassettes had the fixative removed immediately after passage (by passage of filtered air through the cassette until the cassette was free of fixative droplets), were stored overnight at 4 degrees Celsius, and FISH-treated after overnight storage. Other cassettes were stored at 4 degrees Celsius with the fixative retained within the cassette until four or more cassettes had been accumulated, at which time the fixative was removed, the cassettes were stored overnight at 4 degrees Celsius, and the cassettes were FISH-treated following the overnight storage. FISH-treatment entailed removal of the cover and processing using the CEP ® X Spectrum OrangeTM CEP ® Y Spectrum GreenTM as described in Example 3. DAPI was used as a counter-stain and to demonstrate the presence of an intact nucleus.
  • the glass cover having the stained cells attached thereto was examined using a fluorescent microscope either manually or using a computer-controlled stage coupled with an automated detection algorithm.
  • Figure 7 provides a relative "map" of the location of each of the identified cells that provide a positive signal for a male fetal cell. Most of the identified cells are at the exit or outlet portion of the cassette with a few of the cells in the inlet area. This indicates that the cassette is capable of capturing fetal cells and does not permit their passage.
  • Results from one cassette indicated that 1 1 fetal cells (i.e., cells exhibiting fluorescent signals indicative of the presence of both X and Y chromosomes in their nuclei) were captured, as were fewer than about 300 adult female cells (believed to be primarily white blood cells).
  • cassettes were stained with Wright-Giemsa stain to examine the morphology of captured cells. These cassettes were not used for FISH analysis and were observed only by light microscopy. Two of these cassettes were provided to an expert in nucleated white blood cells (a transplantation immunologist) who was not informed as to the nature of the sample that had been applied to the cassettes. This expert opined that the captured cells included an irregular band of predominately "epithelioid cells" having granulocytes and mononuclear cells intermingled therewith.
  • cytological morphology of these cells was described by the expert as epithelial-like, they were believed to be trophoblasts or other large cells, in view of the fact that the immunologist was not told to expect that fetal trophoblasts might be among the cells observed.
  • Fetal trophoblasts are known to be epithelial cells that invade maternal blood vessels in the placenta.
  • cassettes used for the experiments described in this example indicate that the cassette typically captured between about 200 to 4,000 cells from each 1.25-milliliter sample of maternal blood. It is apparent from observations of the captured cells that at least some of the captured cells were cells of fetal origin. However, it is equally apparent that the cassettes are able to capture a variety of other blood-borne cells from blood samples. These other cells include white blood cells. Analysis of the positions at which cells were captured in the cassettes used in these experiments revealed that cells were captured primarily at three distinct regions. Approximately 30-35% of cells were captured at the portion of the cassette at which the steps having cover-to-step distances of 5.2 and 5.4 micrometers occurred.
  • the ratio of neutrophils: monocytes is far higher (1.03 : 1 on the upstream side of the separation chamber and 1.67: 1 on the downstream side) in samples obtained using the devices described herein than the ratio that occurs in normal blood (approximately 50:1).
  • cassette and methods described in this example provide a valuable tool for the selection of cells and other particles of biological interest for therapeutics, diagnostics and general research applications where it is important to either enrich a cell or particle sample for analysis or obtain a pure population for analysis.
  • Applications in genetics, phenotypic analysis epigenetic analysis are areas that could benefit from such isolation processes.
  • Human fetal trophoblasts are believed to exhibit cell diameter generally in the range 14.3 to 30 micrometers.
  • the lumen of mammalian capillaries can exhibit a significantly smaller diameter, on the order of 15 micrometers or smaller (see, e.g., Wang et al., 2007, Exp. Eye Res. 84: 108-117, in which microspheres having a diameter > 8 micrometers administered to arterial blood were observed not to reenter systemic circulation; Maxwell et al, 1985, Heart Circ. Physiol. 248(2):H217-H224 similarly observed a size limit of about 9 micrometers for arterially-administered microspheres passing through intestinal capillary circulation).
  • fetal trophoblasts and similarly large cells in vivo will be selectively concentrated on the arterial side of systemic capillary beds. It follows from this determination that fluids that are on the arterial side of blood capillaries are particularly suitable sources of fetal trophoblasts. Such fluids can include arterial blood, especially arterial blood taken upstream (with respect to
  • capillary beds e.g., pulmonary embolism, pulmonary embolism, and pulmonary embolism.
  • fluids derived from arterial blood prior to passage of the arterial blood through capillaries e.g., lung or bronchial secretions
  • other narrow passages e.g., blood in the common hepatic artery.

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Abstract

L'invention concerne un appareil de ségrégation de particules sur la base de leur capacité à circuler dans un passage à gradins. Au moins certaines des particules demeurent dans un passage limité par un premier gradin, mais au moins certaines de ces particules sont incapables de traverser un passage plus étroit limité par un autre gradin, ce qui crée la ségrégation des particules. L'appareil et les procédés de l'invention peuvent servir à séparer une grande variété de types de particules. Par exemple, il peuvent servir à ségréguer les cellules de type foetal dans un prélèvement de sang maternel, tel qu'un prélèvement de sang artériel maternel.
PCT/US2010/046350 2008-04-23 2010-08-23 Procédés et appareil pour la ségrégation de particules, comprenant des sources supplémentaires de collecte de prélèvements pour la séparation de cellules foetales nucléées WO2011028483A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US12/910,299 US20110065181A1 (en) 2008-04-23 2010-10-22 Methods and Apparatus for Segregation of Particles
US15/586,981 US20170234851A1 (en) 2008-04-23 2017-05-04 Methods and Apparatus for Segregation of Particles
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