WO1996006679A1

WO1996006679A1 - Centrifuge syringe apparatus and method

Info

Publication number: WO1996006679A1
Application number: PCT/US1995/011162
Authority: WO
Inventors: Peter Van Vlasselaer
Original assignee: Activated Cell Therapy, Inc.
Priority date: 1994-08-31
Filing date: 1995-08-31
Publication date: 1996-03-07
Also published as: ATE168288T1; DE69503512T2; HK1013807A1; JPH10509580A; AU680383B2; EP0778794A1; US5577513A; ES2121414T3; AU3502395A; EP0778794B1; JP3487604B2; CA2198606C; DK0778794T3; DE69503512D1; CA2198606A1; NZ292754A

Abstract

Disclosed is a centrifugable cell separation apparatus that is useful in density gradient separation of cells and other biological materials. In a specific embodiment, the apparatus takes the form of a syringe (10) that allows for the withdrawal of a sample and which contains a movable plunger (18) containing a constricted region (28) that restricts mixing between pellet and supernatant fractions following centrifugation. The syringe (10) may also contain a density gradient separation solution (20). Also disclosed are a kit and a method for extracting a fluid specimen that utilise the apparatus.

Description

CENTRIFUGE SYRINGE APPARATUS AND METHOD

1. FTELD OF THE INVENTION

The present invention relates to a centrifugable cell-separation apparatus that is useful in density-gradient separation of cells and other biological materials.

2. BACKGROUND

The prior art contains a number of devices that provide for the extraction of fluid samples as well as their centrifugation. For example, U.S. Patent No. 4,459,997 to Sarstedt discloses a blood extraction and centrifugation device that provides for the withdrawal of blood from a patient into a tube that can be used for centrifugation. The centrifugation tube is a simple straight-walled tube that does not contain a constricted region or provide for the use of density gradient material.

U.S. Patent No. 4,020,831 to Adler discloses a syringe that can draw a specimen, and then allow disassembling of certain parts of the syringe so that the portion of the syringe holding the specimen can be placed in a centrifuge. The syringe also contains a plug of a specific density. During centrifugation, the specimen will separate so that lighter phases are above the plug, and heavier phases are below the plug. This device does not provide for easy removal of the separated phases, and does not provide for the use of a density gradient material.

In addition, U.S. Patent No. 3,965,889 to Sachs discloses an apparatus for the sampling of blood and the separation of plasma. The syringe includes a theπnosealable walled container with a medial restriction into which blood is drawn. After the blood is drawn into the container, the container is removed and placed in a carrier for centrifugation, after which the container can be sealed at the restriction to separate the phases of blood. This device requires the removal of the specimen container to a different carrier for centrifugation, thereby increasing the risk of contamination of the specimen. There is a need in the art for a centrifugation tube that can be used to separate components of a cellular mixture in a manner such that cells present in the supernatant can be readily and quantitatively collected by decantation, without disturbance to or contamination from higher density cells present in the lower phases and the pellet. In particular there is a need for a device mat can be used in conjunction with a density gradient material to effeα separation and collection of relatively rare cells from a mixture. There is also a need in the art for a syringe that can be used to separate materials of different densities which is an integrated unit that does not require transfer of sample to a different container for centrifugation and therefore reduces risk of contamination. The present invention provides these features and a sterile environment in which all required cell-sorting manipulations can be carried out.

3. SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a centrifugable cell-separation apparatus. The apparatus includes a container and a plunger slidably disposed within the container. In a preferred embodiment, the plunger includes a cylindrical housing. In this embodiment, the outer diameter of the housing makes a seal with the inner diameter of the apparatus container.

The container has an orifice that provides for fluid flow into the container. The orifice preferably includes a fitting which allows for sterile transfer of fluid into the apparatus. In preferred embodiments, fluid transfer through the orifice can be introduced by way of a sterile needle or a tubing that is further adapted to connect to a reservoir.

The plunger forms a fluid receiving space. The top wall of the plunger is a constriction member that defines an opening through which fluid flows into the space. The constriction member is constructed in such a way that fluid is retained in the plunger when the plunger is inverted. In a preferred embodiment, the opening defined by the plunger top wall is annular; however, the opening can assume a number of different shapes, including star-shaped, oval, rectangular and the like. Alternatively, the opening can be a plurality of openings or can be covered by a mesh or grid. The apparatus also includes means for sliding the plunger within the apparatus container. In a preferred embodiment, the sliding means is an elongated member secured to the plunger. The elongated member passes through a central orifice in the other end of the container. In a preferred embodiment, the elongated member is removable secured to the plunger bottom wall. In another preferred embodiment, the member may be reattached to the bottom wall.

In a specific embodiment, the invention includes a centrifuge syringe that provides an integral syringe and centrifugation tube in one apparatus and further provides for the use of density gradient material to enhance its cell-separation capabilities. The apparatus has a specimen container with one end having a fitting covering an orifice adapted for the sterile introduction or ejection of fluids, and the opposite end having a central orifice for the sealing engagement with a handle of a plunger. The handle is connected to a plunger at one end, which is located within the container. The opposite end of the handle remains outside the specimen container, and is used to move the plunger longitudinally within the container. In another embodiment, the fluid receiving space of the plunger is filled with a den- sity gradient material. The density gradient material preferably extends to a level above the top wall constriction member, filling part of the upper portion of the container.

In another aspect, the invention includes a closed system for analysis of fluid. Such a system is particularly useful when cells to be separated can be drawn from a patient and directly separated in the apparatus. Alternatively, the cell mixture will be stored in a sterile bag, prior to extraction therefrom and separation by use of the cell separation apparatus. Thus, this embodiment of the invention will include, in addition to the cell separation appa¬ ratus, a fluid sample reservoir, which might include a patient, and tubing sterilely connected between the fluid sample reservoir and the apparatus.

In a further aspect, the present invention will be seen to encompass a kit. The kit includes a cell separation apparatus, as described above, and a quantity of density gradient material sufficient to fill the fluid receiving space in the plunger and to further fill the container to a level above the plunger top wall.

In yet a further aspect, me invention includes a mediod of extracting and centrifu- ging a fluid specimen, to separate components, such as cells, present in the specimen. According to the method, a centrifugation apparatus as described above is filled with a density gradient material to a level above the top wall of the plunger. The specimen sample is then into the apparatus and onto the density gradient material in the apparatus. Centrifugal force is then applied to the apparatus to pull the sample toward the lower end of the apparatus container. The portion of sample remaining above the top wall of the plunger is then removed. Preferably, the desired specimen component will be found in this top fraction; however, the method can also be used to separate sedimenting materials, by further extracting the portion of the separated specimen that remains within the plunger after centrifugation.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross-sectional view of a centrifuge syringe of the invention before the extraction of a specimen;

FIG. 2 shows a cross-sectional view of the centrifuge syringe of FIG. 1 upon introduction of the specimen;

FIG. 3 shows a cross-sectional view of the centrifuge syringe of FIG. 1 after centrifugation and removal of the handle;

FIG. 4 shows a cross-sectional view of the centrifuge syringe of FIG. 1 to which the handle has been re-attached, and from which the specimen has been removed; FIG. 5 is a cross-sectional view of an alternative embodiment of the centrifuge syringe according to the invention;

FIG. 6 is a perspective view of the plunger of the alternative embodiment of FIG. 5;

FIG. 7 is an enlarged view of the plunger of FIG. 5; FIG. 8 is a cross-sectional view of an alternative embodiment of the centrifuge syringe plunger having a valve;

FIGS. 9A-F are cross-sectional views of alternative embodiments of the plunger of the centrifuge syringe;

FIG. 10 is a cross-sectional view of an alternative embodiment of the centrifuge syringe having multiple constriction members; and

FIG. 11 is diagrammatic illustration of a closed system for blood analysis using a centrifuge syringe according to the present invention. 5. DETAILED DESCRIPTION OF THE INVENTION

5.1 Centrifuge-Svrinpe and Methods of Use

The present invention is directed to a centrifugable cell separation apparatus that is particularly adapted to separation of cells from bodily fluids. Generally, the apparatus is a syringe that has a specialized plunger slidably disposed within the syringe chamber. The plunger has a fluid-receiving space into which fluid can be delivered. An important feature of the plunger is that it retains the fluid contained within its fluid receiving space when the syringe is inverted.

During operation, the plunger slides between the two ends of the syringe container or barrel. Such sliding can be effected by conventional methods, such as by attaching to the plunger an elongated member or handle which protrudes through one end of the syringe barrel and applying pressure thereto. Alternatively, it will be understood that the plunger can be made to slide by force, such as fluid force acting on the fluid receiving end of the plunger, or by suction or negative pressure applied to the bottom wall of the plunger. The plunger can also be moved within the syringe barrel by externally applied means, such as by electromagnetic means.

For operation, the plunger is initially disposed in the top portion of the syringe container. As the plunger is drawn toward the bottom of the syringe, fluid is drawn into the syringe and plunger. Alternatively, the plunger, and a portion of the syringe container above the plunger constriction are pre-filled with a cell separation material, such as a density gradient material, and fluid that is drawn into the syringe is layered onto this material. When the syringe is sufficiently filled with material, the handle is removed from the plunger of the syringe for centrifugation. An essentially fluid-tight seal between some portion of the plunger and the inner wall of the centrifugation chamber ensures that sufficiently dense materials in the fluid will flow through the constricted opening in the plunger and pellet in the bottom portion of the plunger.

The foregoing general description of the centrifugable syringe and its method of use is illustrated by the particular embodiments that follow.

5.2. Specific Embodiments

One embodiment of centrifuge syringe 10 according to the invention is illustrated in FIG. 1. Centrifuge syringe 10 includes a specimen container 14 with a central orifice formed by fitting 12 adapted for receiving a needle 13, a handle 16 and a plunger 18. Fitting 12 may be any type of locking tip adapted to hold a needle, for example, a Luer- Lock™ syringe tip. Alternatively, fitting 12 may be a sterile septum adapted for connection with sterile fluid bags and tubes, for example a SAFSITE™ small wire extension set with reflux valve and Spin-Lock™ adaptor available from Burron Medical Inc., Bethlehem, Pennsylvania. Handle 16 further preferably comprises knob 22 and a removable connection 24 to plunger 18. As shown in FIGS. 1-4, plunger 18 is single piece, machined or molded from a plastic material. Known medical grade plastic materials may be used.

Plunger 18 as shown in FIG. 1 has a funnel-shaped bottom wall 26 that is removable connected to the handle at connection 24. Side wall 27 preferably closely fits the container wall to permit sliding movement but provide an essentially fluid-tight barrier therearound. A top wall is formed by constriction member 28, which defines central opening 29. Alternatively, the outer diameter of side wall 27 may be slightly undersized to facilitate sliding and an o-ring seal provided between side wall 27 and container 14. Removable connection 24 may take the form of, for example, a screw fitting or a snap-fit. Preferably, connection 24 also provides for reattachment of handle 16. If reattachment is not desired, connector 24 may be designed such that handle 16 can be broken off. A suitable connection can be selected by those of ordinary skill in the art.

For use in density gradient separation plunger 18 is filled with a cell-separation density-gradient medium 20 before the introduction of a specimen. As is understood by persons of ordinary skill in the art, such materials have specifically defined densities which are selected based on the particular sample material being separated. Examples of cell- separation density-gradient media include sucrose, albumin and Ficoll™. A preferred material is available from Pharmacia Fine Chemicals of Piscataway, New Jersey and Uppsala, Sweden under the trademark PERCOLL™. Preferably, the density gradient material is filled to a level above the constriction member, or at least above the top of opening 29. For example, when using a standard 50ml syringe, having an inner diameter of about 2.8 cm, the gradient material is preferably filled to a level about 1mm or more above constriction member 28. This fill level will help to prevent formation during centrifugation of an interface portion, as explained below, under constriction member 28. Referring to FIG. 2, the introduction of the specimen into centrifuge syringe 10 is illustrated. Specimen 30 is drawn into the syringe through needle 13 secured to fitting 12, aided by the vacuum created by handle 16 and plunger 18 as the handle is pulled out of container 14, drawing the plunger away from fitting 12. The handle should be pulled with sufficiently low force and velocity to avoid mixing of the specimen with the density gradient material onto which the sample is layered. Preferably, when the handle is pulled at an appropriate force, the sample will form a stream which adheres to the side of the container as it is drawn in, as shown in FIG. 2. This will reduce unwanted mixing. Mixing of the two materials is also minimized by the fact that the density of the specimen is preferably significantly lower than the density of the density gradient material. After specimen 30 is drawn into container 14, the container is maintained in an upright position and the sample lies on top of density gradient material 20.

Using needle 13, a sample such as peripheral blood may be drawn directly from a patient for analysis. The present invention thus ensures sterility of such a sample by eliminating direct handling of the sample prior to introduction into the centrifugation container. Alternatively, as illustrated in FIG. 11, using a sterile septum as fitting 12, blood previously collected by known techniques and stored, for example in a sterile bag 33, may be drawn into the centrifugation container through sterile tubing 35 or other known sterile connection means. The present invention thus ensures a sterile transfer of sample material on a larger scale in a completely closed system, again without direct handling of sample material.

Referring again to FIG. 2, once the specimen has been completely drawn into the container 14, and the handle 16 has been pulled so that the removable connection 24 is located at the lower central orifice of the specimen container 14, the handle 16 can be removed for the centrifugation step.

FIG. 3 illustrates the centrifugation syringe after the centrifugation step has been performed. As shown, the handle has been detached from the plunger 18, which is located at the bottom end of the container 14. Centrifugation of container 14 has resulted in a pellet 32 being formed from the heavier portions of the specimen at the bottom of the plunger 18. Density gradient material 20 is located above pellet 32. An interface portion 34, which contains the cells of interest, is formed between specimen diluent 33 and density gradient material 20, and above constriction member 28.

Interface portion 34 may be removed from the centrifuge syringe 10 by reattaching handle 16 to connector 24 and ejecting the interface 37 and supernatant diluent material 33, as well as a portion of density-gradient material 20, as indicated by arrow 37 in FIG. 4. Such ejection may be carried out while the syringe is in an inverted position, as illustrated in FIG. 4, or may be accomplished by ejection in an upright position, to minimize inclusion of density cell separation medium in the ejectate. Alternatively, interface portion 34 can be removed without reattachment of the handle, by opening the syringe below fitting 12, such as at 39, and decanting the supernatant and interface material. Such opening can be accomplished by cutting or by means of an integral fitting in the container of the syringe. Further removal of density gradient material 20 and pellet 32 can be achieved by reattaching handle 16 to plunger 18 at connection 24. The handle then can be pushed into

5 the container to aid the removal of the material if necessary.

According to one theory, the presence of the constriction member with a restricted opening provides a support or nucleus for formation of an intermediate surface tension across the container. This surface tension impedes the mixing of upper and lower regions (above and below the constriction member) of the tube when, for example, the contents of

10 the upper region are ejected from the tube. Accordingly, the dimensions of the opening formed by the constriction member are dictated by the ability to form a surface tension. A constriction member that is little more than a rim around the interior of the barrel may be sufficient to form the necessary surface tension. Hence, the cross-sectional area of the opening formed by the constriction member may be as little as about 5% or as great as

15 about 95% of the horizontal cross-sectional surface area of the syringe. In an exemplary embodiment, where the syringe has an inside diameter of about 2.8 cm, an aperture having a diameter of about 0.5 cm is suitable.

In many applications, it will be desirable to collect only the supernatant fraction containing interface portion 34. In such cases, the pellet is discarded with the syringe. In 0 other cases, the pellet can be removed by mechanical manipulation/disruption. For example, the syringe can be inverted and subjected to vortex mixing. Such mixing will disrupt the pellet into the adjacent liquid phase and will induce movement of this liquid phase and disrupted cells from the second or collection chamber of the syringe into the first chamber of the syringe.

25 An alternative embodiment of the present invention is shown in FIGS. 5-7.

Centrifuge syringe 40 has a plunger 42 formed from separate pieces and without sidewalls. Plunger 42 has a flat bottom plate 44, which may be formed by a washer formed from medical grade plastic such as polycarbonate. Bottom plate 44 is preferably circumscribed by a silicone or rubber seal 46 for the creation of an fluid-tight seal between bottom plate

30 44 and the inside wall of the specimen container 48. Threaded or snap-fit connection 51 is provided in the bottom plate to removable attach handle 50. Plunger 42 has fittings 52, to connect bottom plate 44 to annular constriction member 54, which defines opening 55. Fittings 52 are preferably made of medical grade plastic, such as polycarbonate. Constriction member 54 is funnel-shaped, and preferably made of silicone or rubber. There are preferably three fittings 52, as shown in perspective view of the plunger and handle portion of the device in FIG. 6, but there may be only two, or more than three fittings, if desired. The constriction member can be secured to the fittings by providing stepped recesses 56 in the constriction member, as shown in FIG. 7, for retaining mushroom like- 5 heads 57 on the fittings. Fittings 52 may be glued to bottom plate 44 preferably with medical grade adhesive. Other means for connection may be devised by persons skilled in the art. The particular type of connection used is not critical so long as a secure connection between the parts is maintained.

An advantage of the present invention is that the low density material above the

10 constriction member of the plunger is separated from material beneath by the simple act of ejecting it with the aid of the plunger, as described above. With reference to FIG. 1, if the opening at fitting 12 is large enough or if the container is opened as described with reference to FIG. 3, above, the cells of interest may be poured off. This contrasts with many conventional methods of unloading gradient separations using standard straight-wall

15 centrifuge tubes, where materials are separated by carefully pipetting out of the tube or, alternatively, by puncturing the bottom of the tube and allowing the contents of the tube to slowly drip out into collection vessels. Thus, the present invention provides a convenient, simple means for unloading differentially separated materials. In addition, unlike conventional straight-wall tubes, if the centrifuge syringe is dropped or accidentally

20 inverted, the contents of the upper and lower portions will not readily mix due to the presence of the constriction member. Moreover, once separation has taken place, the solution present above the constriction member can be mixed in the tube, without disturbing (or fear of contamination by) the contents of the syringe below the constriction member. Preferably this is done with the syringe in an inverted position as shown in FIG. 4.

25 The separation of materials may be further enhanced by the addition of valve 60 to the plunger, as shown in plunger 64, illustrated in FIG. 8. Valve 60 is located at opening 62 in plunger 64. Valve 60 may be a one-way valve, or a valve that only opens upon application of a threshold centrifugal force. The valve can be formed by providing flaps of a softer material over hole 62. In a preferred embodiment, the force required to open valve

30 60 would be about 850 times the normal force of gravity. Valve 60 thus allows heavy cells to pass through during initial centrifugation, and then keeps those cells in place, allowing for further processing, such as washing or mixing, of the lighter cells of interest located above the valve. In this way complete and final manipulation of the cells can be performed in a single sterile container. The shape of opening 29, 55 is not limited to a circular shape, though in general a sloped or funnel-shaped constriction member forming a roughly circular shaped annulus will be preferred. The opening may assume other configurations, such as an oval shape, a star shape or other non-circular shape that allows passage of cells through the opening. Alternatively, or in addition, the opening may be formed by a plurality of openings or may be covered by a grid or mesh that allows passage of cells therethrough. Such a mesh or grid arrangement is also referred to herein as a plurality of openings.

FIGS. 9 A-F are illustrations of alternative shapes and designs for the plunger of the centrifuge syringe according to the invention. FIG. 9A shows plunger 70 with a fluid-recei- ving space having a flat bottom wall. FIG. 9B shows plunger 72 with a pointed bottom wall. Plunger 72 with the pointed bottom wall will allow the heavier cells to form a better pellet, which may be desired if the cells are to be collected. Alternatively, plunger 74 with a defined cell-collecting compartment 76 can be utilized to offer collection of cells.

FIG. 9D shows plunger 70 that includes a cell trapping material 78, such as a sponge or gel. Material 78 may contain compounds that specifically bind certain cell types or toxins that kill specific cell types. Material 78 may also be made of a magnetic material if desired.

FIGS. 9E and F show alternative embodiments of the plunger that facilitate movement of the plunger within the container. FIG. 9E shows plunger 80 with extending contact points 82. The plunger 80 will only contact the container at these points. Similarly, in FIG. 9F, plunger 84 is shown with extending contact points 86.

FIG. 10 illustrates a further alternative embodiment of the centrifuge syringe of FIG. 1 with an additional constriction member. Dual constriction syringe 90 has a bottom plate 92 connected to a first constriction member 94 by fittings 96. Second constriction member 98 is located above first constriction member 94 to create an additional compartment, to allow separation of cells of differing densities. Second fittings 97 may be used to secure second constriction member 98. Additional constriction members could also be added if a sample of several different densities is to be separated.

FIG. 10 also illustrates one embodiment of the removable and re-attachable connection means between the handle 102 and the bottom plate 92. In this embodiment, an internal screw 100 forms the attachment means between the handle and the bottom plate, so that the handle 102 can be removed and then reattached after centrifugation.

Preferably, the centrifugation syringe according to the present invention would be provided as a sterilized complete unit with the density gradient material already in place to an appropriate level. In this way, sterility of the syringe is guaranteed and the user need only open the sterile packaging to use the invention. Alternatively, the syringe can be provided in kit form with the density gradient solution separately provided and the needle and handle disattached. The user would then fill the plunger of the syringe with density gradient material, and then assemble the needle and handle before use.

6. EXAMPLES

The following examples illustrate, but in no way are intended to limit the present invention.

EXAMPLE 1 MATERIALS FOR ENRICHMENT OF CD34* CELLS FROM BLOOD CELL

MIXTURE

1. Peripheral Blood and Bone Marrow

Patients were hydrated and treated with cyclophosphamide (4 gm/m²) administered by intravenous (IV) infusion over two hours through a central venous catheter. Twenty- four hours after the completion of the cyclophosphamide infusion, patients were treated with G-CSF (NEUPOGEN, Amgen, Thousand Oaks, CA) administered by subcutaneous (SC) in- jection at a dose of approximately 10 μg/kg/d. Apheresis was initiated upon recovery of the white blood cell count (WBC) to equal or more than 1 x lO'/L. Apheresis was per¬ formed using a Cobe Spectra Cell Separator (Lakewood, Colorado) at a rate of 80 ml/mln for 200 min (total volume of 16 L).

Apheresed peripheral blood was applied directly onto the density gradient. However, complete blood and bone marrow aspirates were processed to a buffy coat (removal of red cells) before they were applied onto the density gradient.

2. Preparation of Density Gradients "PERCOLL" solution was purchased from Pharmacia Biotech (Uppsala, Sweden) and stored at 4°C according to the recommendation of the vendor. A stock solution was prepared by mixing 12 parts of "PERCOLL" with 1 part of 10 x calcium and magnesium- free phosphate buffered saline (PBS). The pH of the solution was adjusted to 7.4 and the osmolality to 280 mOsm/Kg H₂O. For use in separating CD34* cells in a cell mixture, the stock solution was further diluted with calcium and magnesium-free PBS to a density of 1.0605 +. 0.0005 gr/ml and used at room temperature. By adjusting the density of the gradient to an accuracy of within +0.0005 gr/ml of 1.0605 gr/ml, reproducibility and accuracy of cell separation was ensured. This was done using a high precision digital density meter such as DMA 48 (Anton PAAR USA, Ashland, VA). All procedures were performed under sterile conditions and at room temperature.

EXAMPLE 2 ISOLATION OF CD 34* PROGENITOR HEMATOPOIETIC CELLS USING CENTRIFUGE SYRINGE

The centrifuge syringe and the method of the invention can be used to isolate CD34⁺ progenitor cells from patients treated with chemotherapy and granulocyte colony stimulating factor (G-CSF) as described in Example 1 above. These cells can then be used to repopulate the patient's lymphohematopoietic system. Human peripheral blood mononuclear cells (PBMC) are obtained by apheresis of patients treated with daily injections of G-CSF (lOμg/kg/day). Samples are then processed according to standard methods understood by persons skilled in the art.

Cells are resuspended in 25 ml of calcium-free, magnesium-free PBS and then drawn into the syringe on top of 15 ml of PERCOLL™ solution in a 50 ml conical centrifuge syringe fitted with a plunger containing a constriction member, as illustrated in FIG. 1. This PERCOLL™ solution has a density of 1.0605 g/ml (osmolality 280±5 mOsm/kg H₂O; pH 7.4). The diameter of the opening in the constriction member of the syringe preferably is about 0.5 cm. This volume of PERCOLL™ is sufficient volume to fill the container to a level higher than about 1mm above the constriction member. After the sample is drawn in, the needle and plunger are detached. The centrifuge syringe is then centrifuged at about 850xg for 30 minutes at room temperature. The upper fraction containing CD34⁺ cells is collected by ejecting the sample into a sterile container.

Cell type and purity in the collected fraction are tested according to standard methods to determine enrichment of functional CD34⁴ cells. For example, cells can be tested for presence of colony forming units (CFU; indicating committed hematopoietic progenitor cells), Long term culture initiating cells (LTC-IC; indicating uncommitted hematopoietic progenitor cells), natural killer (NK) cells, and natural suppressor cell activity in the interface fraction, according to methods known in the art. Using the apparatus and method described above, the interface contains approximately 70-90% of the CD34⁺ cells and more than 90% of the CFU's.

While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.

Claims

WHAT IS CLAIMED IS:

1. A centrifugable cell-separation apparatus, comprising: a container having side walls, a first end and a second end, said first end defining an orifice adaptable to provide a sterile connection for fluid flow therethrough, a plunger slidably disposed within said container, said plunger having a bottom wall joined to a top wall defining therebetween a fluid receiving space, said top wall defining a constriction member positioned and constructed to define an opening to receive fluid and to retain fluid in said fluid receiving space, when the plunger is inverted, and means for sliding said plunger between said first and second ends of said container.

2. The cell separation apparatus of claim 1, wherein said means for sliding said plunger comprises an elongated member secured to said plunger and passing through a central orifice defined by said second end of said container.

3. The cell separation apparatus of claim 1 or 2, wherein said orifice defined by said first end includes a fitting adapted to provide a sterile connection for fluid flow therethrough, and said constriction member is an annular member defining a central opening.

4. The cell separation apparatus of claim 3, further comprising a hollow needle secured to the fitting for flow of a fluid sample therethrough.

5. The cell separation apparatus of claim 3, further comprising a sterilizable tubing secured to the fitting for flow of a fluid sample therethrough, said tubing being adapted for communication with a sterile fluid sample container.

6. The cell separation apparatus of any of claims 1, 2 or 3, further comprising cell-separation medium disposed within said fluid receiving space and extending to a level above said constriction member in the container.

7. The cell separation apparatus of claim 2 or 3, wherein said elongated member comprises a substantially rigid handle removable secured to the plunger bottom wall.

8. The cell separation apparatus of claim 7, wherein the handle reattachably secures to the plunger bottom wall.

9. The cell separation apparatus of claim 1 or 2, wherein said plunger includes a cylindrical housing that has an outer diameter which sealingly engages with the inner diameter of said container.

10. The cell separation apparatus of claim 1 or 2 wherein said opening defined by said constriction member in said apparatus comprises a plurality of openings.

11. A closed system for centrifugation analysis of fluid, comprising any of the cell separation apparatuses defined by claims 1-10, and which further includes: a fluid sample reservoir, tubing connected between said fluid sample reservoir and said first end of said cell separation apparatus for flow of fluid from said reservoir into said apparatus.

12. A centrifugation kit, comprising any of the cell separation apparatuses of claims 1-10, and a quantity of cell-separation medium sufficient to fill the fluid receiving space in the plunger and extend to a level in the container above the plunger top wall.

13. A method of extracting and centrifuging a fluid specimen comprising the steps of: providing a centrifugation apparatus defined by any of claims 1-10; filling the fluid receiving space in the plunger of the apparatus with cell-separation medium to a level above the top wall of the plunger; drawing a sample into the apparatus and onto the cell-separation medium; applying centrifugal force to said apparatus such that sample in said apparatus is pulled toward the second end of the container; and removing a portion of the sample remaining above die top wall of said plunger after applying centrifugal force to said apparatus.