Patent ID: 12220512

DETAILED DESCRIPTION OF THE INVENTION

One variation of the apparatus is illustrated in the perspective view ofFIG.1which shows assembly10which may be used to effectively concentrate one or more plasma constituents and/or cells from blood plasma or PRP without requiring centrifugation. The assembly10may concentrate the constituents and/or cells with or without white blood cells. As shown, a first reservoir or syringe22may be fluidly coupled to a second reservoir or syringe26via a manifold12which also provides receiving channels to temporarily or permanently secure the tips of the respective syringes22,26to the manifold12. While syringes are shown and described, alternative fluid container or reservoir configurations may be used instead. Each of the syringes22,26may be integrated directly with the manifold12or optionally fitted with non-standard couplings to ensure that one or both syringes22,26cannot be inadvertently detached or attached to an incorrect port.

A fluid channel16may extend through the manifold12to fluidly couple the openings of the first syringe22and second syringe26and a withdrawal channel18may extend through the manifold12to fluidly couple to the fluid channel16which may have a diameter of, e.g., 5 mm, but may range anywhere from, e.g., 3 to 10 mm. An actuatable valve20which may be configurable between an open and closed configuration, e.g., automatically or manually via a stopcock, may be incorporated with the withdrawal channel18to prevent the release of fluid from the fluid channel16. A filter or frit14may also be incorporated into the manifold12at the entry of the withdrawal channel18to filter select components from passing into the withdrawal channel18from the fluid channel16.

The fluid channel16may optionally incorporate a number of projections or features such as vanes, spikes, crosshairs, etc., to facilitate the effective mixing of fluids as they pass through the fluid channel16. Additionally, a volume of desiccating beads or elements may also be added within the fluid channel16or directly within one or both of the syringes22,26. With the volume of desiccating beads or elements included within the fluid channel16or directly within one or both of the syringes, the device be optionally tapped or shaken or otherwise moved while being held in a horizontal configuration in order to facilitate the dispersion of the volume of desiccating beads over the bottom of the connecting channel16or within the syringes22,26before introducing the material to be concentrated. Doing so may reduce the likelihood of forming any clumps immediately upon introduction of the plasma or PRP into contact with the beads.

A volume of blood plasma or PRP to be concentrated may be introduced within the first syringe22or second syringe26(or both) and the syringes22,26may be coupled to their respective ends in the manifold12such that their openings are positioned at each end of the fluid channel16. With the volume of desiccating beads retained within the fluid channel16and/or within one or both syringes22,26, the respective plungers24,28may be actuated in an alternating manner to push the blood plasma or PRP back-and-forth between the syringes22,26through the fluid channel16. As the blood plasma or PRP is cycled between the syringes22,26, the withdrawal channel18may remain closed to prevent leakage from the fluid channel16. As the blood plasma or PRP cycles between the syringes22,26through the fluid channel16, it may become concentrated as the volume of beads mixes thoroughly with the blood plasma or PRP and absorbs various components within the blood plasma or PRP such as water, electrolytes and small proteins, thus leaving a platelet-rich plasma concentrate. Because the mixture of the beads and blood plasma or PRP is cycled back-and-forth between the syringes22,26, the syringe connections to the manifold12may be integral to ensure a fluid-tight connection and the internal diameter of the syringe couplings may be tapered internally at their proximal ends, e.g., to a diameter (2 to 6 mm) relatively smaller than the syringe bore but relatively larger than a standard syringe barrel bore to reduce the likelihood of clogging due to any bead clumps which may be formed by gel polarization.

Once the blood plasma or PRP has been sufficiently concentrated, the valve20along the withdrawal channel18may be opened to allow for the withdrawal of the concentrated product as it passes through the fluid channel16. The filter or frit12may prevent the passage of the beads or other components into the withdrawal channel18thus allowing only the concentrated product to pass through withdrawal channel18and into a reservoir such as a collection syringe. The withdrawal channel18may be opened to allow for the removal of the concentrated product as the bead and blood plasma or PRP mixture continues to cycle through the fluid channel16. This ensures that the concentrated product may be harvested uniformly from the mixture. Also, sweeping of the bead surfaces during product extraction further ensures that if the beads are not yet fully saturated, any residual water absorption does not cause gel polarization and loss of macromolecular constituents or clumping.

Alternatively, both plungers24,28in each respective syringe22,26may be simultaneously pressed to create pressure to force the concentrated product from both syringes22,26and through the withdrawal channel18for collection with the valve20opened. In any case, the filter or frit14may prevent the beads from passing into the withdrawal channel18so that only the concentrated product may pass through.

In an alternative variation, an additional withdrawal channel and port may be provided so that the material can be introduced without having to pass through the filter or frit14. As the material is introduced through the manifold12, it may mix with the beads contained within and sweep them into one or both of the syringes22,26forcing the respective plungers24,28to extend partially.

These beads may function as a concentrator when contacting and mixing with the blood plasma or PRP and may comprise any number of materials which are configured to selectively absorb particular components such as water, electrolytes, small proteins, etc. leaving a concentrated product for collection. Examples of various materials that the beads may be fabricated from may include any suitable material for processing the biological materials such as various polymers, metals, minerals, polysaccharides, silica gel, ceramics, glasses, etc.

Various examples of polymers may include, e.g., dextranomer, polystyrene, polyethylene, polyvinyl chloride, polypropylene, polyacrylamide, etc. Various examples of metals may include, e.g., titanium, etc., while various examples of minerals may include, e.g., zeolites, corundum, quartz, etc. Various examples of polysaccharides may include, e.g., alginate gel, starch, dextran, agarose, etc.

In some variations, the beads may be conjugated with an activating material such as an antibody such as immunoglobulin g.

Because the beads are used to concentrate the blood plasma or PRP by inducing a change in the biological material, the size and composition of the beads can be varied depending upon the bead materials used and the biological material to be concentrated. In one example, the beads may be comprised of a polyacrylamide material (e.g., Bio-Rad P6 chromatography beads having a nominal molecular weight limit (NMWL) of 1,000-6000) and may be sized to each have a dry bead diameter of, e.g., 45 to 90 micron. Hence, for a given volume of blood plasma or PRP to be concentrated such as about 10 ml, although the volume may range anywhere from, e.g., 5 to 30 ml, the amount of beads used may be about 1,000 mg although the amount may range anywhere from, e.g., 200 to 5,000 mg, in weight or at ratio of beads to blood plasma or PRP may be about 0.1 but may range anywhere from, e.g., 0.04 to 0.15, to effectively separate out the desired components.

Furthermore, depending upon the beads and the biological material to be concentrated, the number of back-and-forth cycles between the syringes22,26can vary. In one variation, the blood plasma or PRP may be cycled, e.g., 10 to 50 cycles, to sufficiently mix the beads with the blood plasma or PRP. Alternatively, the cycling may be timed to allow for sufficient mixing and may range from, e.g., 30 sec. to 2 min. or longer. Additionally, with the diameter of the fluid channel16being relatively small relative to the size of the beads, any clumps which may form due to imperfect mixing may be broken apart by turbulence and shear as the clumps pass through the channel16. Moreover, the method of cycling back-and-forth between the syringes22,26facilitates an efficient mixing of beads with the blood plasma or PRP to produce a more uniform slurry with relatively less frothing.

FIGS.2A to2Dshow perspective, side, bottom, and top views, respectively, of another manifold variation which may be used with conventional syringes or modified syringes. The manifold30may include a manifold body32which includes respective ports34,36positioned on opposite ends of the manifold body32for fluidly connecting to one another via fluid channel38extending through the manifold body32and between the ports34,36. A withdrawal channel40may extend from the fluid channel38through a filter or fit42positioned to prevent or inhibit any beads or other components from passing through and into the withdrawal channel40.

Although the withdrawal channel40is shown extending transversely relative to the fluid channel38, the withdrawal channel40may be angled at a non-normal angle relative to the fluid channel38instead. Moreover, the assembly30may be used with conventional syringes while in other variations the ports34,36may be keyed or modified to engage temporarily or permanently with specially configured syringes.

Example 1

In one example of a test which was performed, a first volume50A of citrated bovine PRP was introduced directly into the first syringe22which was coupled to port36and the withdrawal channel40outlet was sealed via a cap, as shown in the perspective view ofFIG.3A. A second syringe26with its plunger28fully depressed was coupled to the port34and a volume of concentrating beads52was preloaded into the fluid channel38contained within manifold body32prior to attaching the PRP-loaded first syringe22.

The first plunger24was depressed forcing the first volume50A of PRP to pass into the fluid channel38and into contact with beads52contained within. As the first volume50A is further introduced, the PRP and beads may begin mixing as they are swept into the opposed second syringe26to capture a second volume50B while forcing the second plunger28to expand, as shown in the perspective view ofFIG.3B.

The process of gently cycling the PRP back-and-forth between the syringes22,26through the manifold body32may be maintained for a period of time, e.g., about 30 sec., to allow for the beads52to sufficiently absorb water from the plasma. Once the mixing is completed, the resulting concentrated mixture may appear as a uniform slurry with no bead clumps visible. The cap (or valve, if present) may be removed and a collection syringe54may be attached to the withdrawal port40with its plunger fully depressed, as shown in the perspective view ofFIG.3C. The cycling may be continued between the first and second syringes22,26while the concentrated product56may be forced to pass through the filter or frit42, which prevents the beads from passing, by the modest pressure generated by the flow restriction and resistance of the syringes22,26. This pressure may force the concentrated PRP through the bead-retaining filter or frit42and into the collection syringe54, as shown in the perspective view ofFIG.3D.

When the bulk of concentrated fluid56had been expressed through the filter or fit42into the collection syringe54, the remaining bead pack52may appear uniform and white, indicating that the mixing and harvesting had maintained uniformity of the slurry and that concentrated fluid56had been almost entirely transferred out into the collection syringe54, as shown by the perspective view ofFIG.3E.

Example 2

In another example of a test which was performed, a volume of buffy coat60A comprised of plasma, platelets, white blood cells, and a small amount of red blood cells was introduced into the first syringe22which was coupled to port36opposite to the second syringe26which was coupled to port34with its plunger28fully depressed, as shown in the perspective view ofFIG.4A.

An amount of the beads52was preloaded into the first syringe22with the volume of buffy coat60A although the beads52may be alternatively preloaded within the fluid channel38of the manifold body32. The volume of buffy coat was then cycled between the syringes22,26through the fluid channel38to thoroughly mix the beads52between the volumes60A,60B, as shown in the perspective view ofFIG.4B. In order to prevent clumping of the beads52(such as when first contacted by the fluid), the volume may be quickly cycled between the syringes22,26particularly when the beads52are preloaded directly within one of the syringes.

After a period of time, e.g., about 30 seconds, of cycling the mixed slurry back-and-forth between the syringes22,26, a collection syringe62was attached to the outlet port40and concentrated product64was expressed though the bead-retaining filter or frit42into the collection syringe62by continuing to pass the slurry back-and-forth between the in-line syringes22,26, as illustrated in the perspective view ofFIG.4C.

After concentrated product64was collected, the remaining bead pack52may appear uniform and slightly pink in color from traces of red blood cells in the small remaining volume of fluid interstitial between the packed beads52.FIG.4Dillustrates a perspective view showing how the concentrated product64is collected while the beads52may remain within the first syringe22and/or second syringe26.

As shown by both examples above, in concentrating PRP and the buffy coat, the ratio of the volume recovered to initial input volume was consistent with the amount of water expected to be absorbed by the amount of beads52employed. This indicated an effective concentration of cells and macromolecules. In separate preliminary testing, cell counts were conducted and confirmed that cell recoveries were relatively high.

Alternative Configurations

A number of optional configurations are possible, for example, one of the two in-line syringes may be fitted with a biasing element such as a spring so that mixing can be effectuated by alternately applying and releasing pressure on only one syringe. The two syringes need not be parallel, but may be connected via a connecting manifold which adjoins them at any angle relative to each other. The perspective view ofFIG.5shows a syringe22coupled to a manifold body70containing a filter or frit72. The second modified syringe78may be coupled to the opposing port of the manifold body70but rather than having a plunger, the second syringe78may contain the biasing element80within and anchored between the movable plunger and an anchoring platform82which may be securely positioned relative to the body of the syringe78.

The biasing element80may maintain the plunger in a compressed state such that the plunger is biased to push away from the anchoring platform80. With this configuration, the first syringe22may contain the beads52, as shown, or the manifold body70may contain the beads52. In either case, the blood plasma or PRP may be introduced into the first syringe22and the plunger24may be depressed to force the mixture of the beads52and blood plasma or PRP through the fluid channel of the manifold body70and into the second syringe78. The plunger24of the first syringe24may be released to allow for the biasing element80to automatically push the plunger within the second syringe78to force the mixture back through the manifold body70and back into the first syringe22. This process may be repeated, as described herein, until the fluid has been sufficiently concentrated. The stop cock76or valve (if present) may be opened to allow for the concentrated product to pass through the filter or frit72and through the withdrawal channel74and into the attached collection syringe86.

In yet another embodiment as shown in the perspective view ofFIG.6, a volume of blood plasma or PRP may be concentrated by connecting a first syringe22to a second syringe88via a manifold70containing a filter or frit72, as previously described. The first syringe22may be optionally preloaded with a volume of beads52while the second syringe88may contain a volume of blood plasma or PRP89to be concentrated. In other variations, the volume of beads52may instead be preloaded within the manifold70itself. The manifold70or the second syringe88may include a valve91such as a stopcock which may be initially closed to prevent communication between the first and second syringes22,88when initially coupled to the manifold70.

With the plunger of the first syringe22initially fully depressed, the plunger may be pulled proximally with the valve91closed such that a vacuum force is created within the first plunger22. This also allows for the volume of beads52contained within to be dispersed within the syringe22prior to contacting the fluid contained within the second syringe88. The first plunger may be locked into its retracted position via a locking mechanism93and the valve91may be opened so that the vacuum within the first syringe22may pull the fluid89in through the manifold70from the second syringe88. This sudden dispersion of fluid89into the first syringe22may help to minimize any likelihood of clumping of the beads52within the first syringe22upon initial fluid contact and may also facilitate the rapid introduction of the fluid89after the valve91is opened.

Once the fluid89has been introduced into the first syringe22, the locking mechanism93may be released (e.g., by twisting the plunger rod relative to the syringe body) and the cycling of the slurry between the two syringes22,88may be accomplished. A collection syringe may be subsequently coupled to the manifold70for withdrawal, as previously described. Similarly, the syringes22,88may be attached via a straight connector or a connector which adjoins the two syringes at any angle relative to each other.

FIGS.7A and7Bshow partial cross-sectional side and perspective views of a manifold90which allows for the two syringes to be fluidly coupled to one another at various angles. As shown, the manifold body92may have a first port96and a second port98both positioned along a first surface of the manifold92such that the ports96,98are in proximity to one another and fluidly connected via curved or angled fluid channels100,102. An optional chamber94may be incorporated between the fluid channels100,102for holding a volume of beads52within during the initial mixing phase between the syringes. Additionally, a filter or frit well104may also be defined to extend from the chamber94for retaining the frit within the chamber94as well as allowing for a withdrawal syringe to be coupled to the manifold body92.

Because the ports96,98are located along a common surface of the manifold body92, the syringes may be angled relative to one another and relative to the manifold body92in any number of angles. Furthermore, this variation may be utilized with any of the devices and methods described herein.

Additionally, in any of these variations described herein, the product collection syringe (or other collection vessel, such as a centrifuge tube integrating a frit above a distal volume of receiving space) could be subjected to centrifugation to centrifugally drive the concentrate through the fit, separating it from the bead pack. This may have the advantage of improving volume recovery, by driving all interstitial fluid between the beads out of the bead pack although this would required an extra transfer step and a centrifuge.

Yet another alternative to pressure applied to the slurry for recovering the concentrated product is to blow the concentrated product out of the bead pack through a fit with, e.g., a steam of air. This affords a good volumetric recovery requires a source of pressurized air. Furthermore, it may also result in frothing and potential damage to cells and plasma proteins if not done with care.

With regard to volume recovery, it should be noted that with close packing of the beads, about ⅓ of the total pack volume is comprised of interstitial space. By squeezing out the concentrated fluid under pressure, this volume may not be recoverable. Regardless of the method (pressure or centrifugation) used to recover the concentrate, fold concentration may be limited to about 3 times due to the difficulty of effectively mixing a slurry comprising a volume ratio of (fully swollen) beads to residual interstitial fluid greater than two. A reasonable volumetric recovery of product harvested by pressure may be on the order of, e.g., 1.5 to 2 times.

In yet another variation for recovering a volume of concentrated product, the coupling110shown inFIG.8may be used in combination with a centrifuge rather than withdrawing the concentrated product directly from the manifold. The coupling110may have a housing112with an introduction port114, such as a Luer connector, positioned upon an upper portion of the housing112into which a mixed slurry may be introduced. A withdrawal port116, such as a Luer connector, may be positioned upon a lower portion of the housing112and a fit118may be positioned within the housing112to separate the internal volume into an upper portion120and a lower portion122.

With the mixed slurry introduced through port114and into the upper portion120of housing112, both ports114,116may be capped or closed and entire coupling110may undergo centrifugation to drive the fluid from the upper portion120, through the fit118, and into the lower portion122of housing112. Once all concentrated product has been separated into the lower portion122and the beads remaining within the upper portion120above the fit118, the withdrawal port116may be opened to withdraw the concentrated product from the housing112.

In one example of use for the housing120, a manifold12may be coupled to respective syringes22,26to initially mix the volume of blood plasma or PRP with a volume of desiccating beads which may freely intermix with the volume of blood plasma or PRP as it is passed between the syringes22,26, as described above and as shown inFIG.9A. The resulting slurry or mixture130of blood plasma or PRP and desiccating beads may be transferred to a single syringe26which may be decoupled from the manifold12and then fluidly coupled to the introduction port114. The slurry or mixture130from the syringe26may then be transferred through the introduction port114and into the upper portion120of housing112, as shown inFIG.9B.

Once the slurry or mixture130has been sufficiently transferred, the syringe26may be decoupled from the introduction port and housing112may then undergo centrifugation to drive the slurry or mixture130from the upper portion120, through the frit118, and into the lower portion122of housing112. The frit118may retain the desiccating beads having absorbed one or more components from the blood plasma or PRP such that the resulting concentrated product134may be retained within the lower portion122. An additional syringe132may be fluidly coupled to the withdrawal port116to then withdraw the concentrated product134from the lower portion122and into the additional syringe132, as shown inFIG.9C.

The apparatus and methods disclosed above are not limited to the individual embodiments which are shown or described but may include combinations which incorporate individual features between the different variations. Modification of the above-described assemblies and methods for carrying out the invention, combinations between different variations as practicable, and variations of aspects of the invention that are obvious to those of skill in the art are intended to be within the scope of the claims.