PLASMA/CELL CONCENTRATOR APPARATUS AND METHODS
20210330873 · 2021-10-28
Assignee
Inventors
- Randy Dorian (San Diego, CA, US)
- Michael D. Leach (Warsaw, IN, US)
- Richard W. Storrs (Berkeley, CA, US)
Cpc classification
B04B2005/0485
PERFORMING OPERATIONS; TRANSPORTING
B01L2400/0622
PERFORMING OPERATIONS; TRANSPORTING
B04B2005/0478
PERFORMING OPERATIONS; TRANSPORTING
B01L3/5635
PERFORMING OPERATIONS; TRANSPORTING
B01L2200/16
PERFORMING OPERATIONS; TRANSPORTING
A61M1/3486
HUMAN NECESSITIES
B04B5/0442
PERFORMING OPERATIONS; TRANSPORTING
International classification
A61M1/34
HUMAN NECESSITIES
Abstract
Plasma/cell concentrator apparatus and methods are described herein for concentrating constituents from a fluid. Generally, a volume of the fluid may be urged from a first reservoir to a second reservoir through a fluid channel and a volume of desiccant for mixing with the fluid may be introduced to create a mixture. This fluid and desiccant mixture may be passed between the first and second reservoirs until one or more components from the fluid are absorbed by the desiccant. After mixing, the fluid may be withdrawn through a withdrawal channel which is in fluid communication with the fluid channel while preventing the desiccant from passing into the withdrawal channel.
Claims
1. An apparatus for concentrating constituents from a fluid, comprising a connector having a first port and a second port connected via a fluid channel; a first reservoir having a variable pressure attachable to the first port; a second reservoir having a variable pressure attachable to the second port; a withdrawal channel in fluid communication with the fluid channel; and a filter positioned to prevent passage of a desiccant from the fluid channel into the withdrawal channel.
2. The apparatus of claim 1 wherein the connector comprises a manifold body.
3. The apparatus of claim 1 wherein the first reservoir and second reservoir each comprise a syringe having a plunger.
4. The apparatus of claim 1 further comprising a valve in communication with the withdrawal channel.
5. The apparatus of claim 1 wherein the filter comprises a frit.
6. The apparatus of claim 1 further comprising a volume of desiccant beads for introduction into the first or second reservoir.
7. The apparatus of claim 1 wherein the second reservoir further comprises a biasing element positioned to provide a pressure against a plunger within the second reservoir.
8. The apparatus of claim 1 wherein the first reservoir further comprises a locking mechanism configured to maintain a position of a plunger within the first reservoir to create a vacuum force.
9. A method for concentrating constituents from a fluid, comprising: urging a volume of the fluid from a first reservoir to a second reservoir through a fluid channel; passing the fluid and a volume of desiccant between the first and second reservoirs until one or more components from the fluid are absorbed by the desiccant; and withdrawing the fluid through a withdrawal channel which is in fluid communication with the fluid channel while preventing the desiccant from passing into the withdrawal channel.
10. The method of claim 9 wherein the volume of fluid comprises blood plasma or PRP.
11. The method of claim 9 wherein urging the volume of the fluid comprises passing the volume from a first syringe to a second syringe through the fluid channel defined through a manifold.
12. The method of claim 9 wherein passing the fluid and the volume of desiccant comprises introducing desiccant beads.
13. The method of claim 9 wherein passing the fluid and the volume of desiccant comprises passing the fluid for a period of 30 sec. to 2 min.
14. The method of claim 9 wherein withdrawing the fluid comprises releasing a valve such that the withdrawal channel comes into fluid communication with the fluid channel.
15. The method of claim 14 wherein preventing the desiccant from passing comprises passing the fluid through a filter or frit such that the desiccant is inhibited from entering into the withdrawal channel.
16. The method of claim 9 further comprising collecting the fluid from the withdrawal channel via a collection syringe.
17. The method of claim 9 wherein withdrawing the fluid comprises withdrawing from the withdrawal channel while the fluid and desiccant are passed between the first and second reservoirs.
18. The method of claim 9 wherein withdrawing the fluid comprises withdrawing from the withdrawal channel by inducing a pressure into each of the first and second reservoirs.
19. The method of claim 9 wherein the second reservoir comprises a biasing member contained within which provides a biasing force against a plunger within the second reservoir.
20. The method of claim 9 wherein urging the volume of the fluid comprises inducing a vacuum within the second reservoir to draw the volume into the first reservoir.
21. The method of claim 9 wherein urging the volume of fluid further comprises retaining the desiccant within a manifold between the first reservoir and the second reservoir prior to passing the fluid and the volume of desiccant between the first and second reservoirs.
22. The method of claim 9 wherein the volume of fluid further comprises retaining the desiccant within the first or second reservoir prior to passing the fluid and the volume of desiccant between the first and second reservoirs.
23. An apparatus for concentrating constituents from a fluid, comprising: a housing defining a first portion and a second portion within; a filter positioned within the housing and separating the first portion and the second portion; a first port in fluid communication with the first portion; a second port in fluid communication with the second portion, wherein the first portion is configured to receive through the first port a mixture comprised of a fluid and a desiccant such that imparting a centrifugal force upon the housing urges the mixture through the filter and into the second portion, and wherein the desiccant having one or more components absorbed from the fluid is retained by the filter and a concentrated fluid is urged into the second portion.
24. The apparatus of claim 23 wherein the first portion comprises an upper portion within the housing and the second portion comprises a lower portion within the housing.
25. The apparatus of claim 23 wherein the filter comprises a frit configured to retain the desiccant.
26. A method for concentrating constituents from a fluid, comprising: introducing a mixture comprised of a fluid and a desiccant into a first portion of a housing; imparting a centrifugal force upon the housing such that the mixture is urged to pass through a filter positioned between the first portion and a second portion of the housing; and receiving a concentrated fluid into the second portion whereby the desiccant having one or more components absorbed from the fluid is retained by the filter.
27. The method of claim 26 further comprising withdrawing the concentrated fluid through a withdrawal port in fluid communication with the second portion.
28. The method of claim 26 wherein introducing the mixture comprises introducing the mixture through an entry port in fluid communication with the first portion.
29. The method of claim 26 wherein the mixture is urged to pass through a filter comprising a frit.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0029]
[0030]
[0031]
[0032]
[0033]
[0034]
[0035]
[0036]
[0037]
DETAILED DESCRIPTION OF THE INVENTION
[0038] One variation of the apparatus is illustrated in the perspective view of
[0039] A fluid channel 16 may extend through the manifold 12 to fluidly couple the openings of the first syringe 22 and second syringe 26 and a withdrawal channel 18 may extend through the manifold 12 to fluidly couple to the fluid channel 16 which may have a diameter of, e.g., 5 mm, but may range anywhere from, e.g., 3 to 10 mm. An actuatable valve 20 which may be configurable between an open and closed configuration, e.g., automatically or manually via a stopcock, may be incorporated with the withdrawal channel 18 to prevent the release of fluid from the fluid channel 16. A filter or frit 14 may also be incorporated into the manifold 12 at the entry of the withdrawal channel 18 to filter select components from passing into the withdrawal channel 18 from the fluid channel 16.
[0040] The fluid channel 16 may 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 channel 16. Additionally, a volume of desiccating beads or elements may also be added within the fluid channel 16 or directly within one or both of the syringes 22, 26. With the volume of desiccating beads or elements included within the fluid channel 16 or 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 channel 16 or within the syringes 22, 26 before 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.
[0041] A volume of blood plasma or PRP to be concentrated may be introduced within the first syringe 22 or second syringe 26 (or both) and the syringes 22, 26 may be coupled to their respective ends in the manifold 12 such that their openings are positioned at each end of the fluid channel 16. With the volume of desiccating beads retained within the fluid channel 16 and/or within one or both syringes 22, 26, the respective plungers 24, 28 may be actuated in an alternating manner to push the blood plasma or PRP back-and-forth between the syringes 22, 26 through the fluid channel 16. As the blood plasma or PRP is cycled between the syringes 22, 26, the withdrawal channel 18 may remain closed to prevent leakage from the fluid channel 16. As the blood plasma or PRP cycles between the syringes 22, 26 through the fluid channel 16, 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 syringes 22, 26, the syringe connections to the manifold 12 may 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.
[0042] Once the blood plasma or PRP has been sufficiently concentrated, the valve 20 along the withdrawal channel 18 may be opened to allow for the withdrawal of the concentrated product as it passes through the fluid channel 16. The filter or frit 12 may prevent the passage of the beads or other components into the withdrawal channel 18 thus allowing only the concentrated product to pass through withdrawal channel 18 and into a reservoir such as a collection syringe. The withdrawal channel 18 may 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 channel 16. 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.
[0043] Alternatively, both plungers 24, 28 in each respective syringe 22, 26 may be simultaneously pressed to create pressure to force the concentrated product from both syringes 22, 26 and through the withdrawal channel 18 for collection with the valve 20 opened. In any case, the filter or frit 14 may prevent the beads from passing into the withdrawal channel 18 so that only the concentrated product may pass through.
[0044] 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 frit 14. As the material is introduced through the manifold 12, it may mix with the beads contained within and sweep them into one or both of the syringes 22, 26 forcing the respective plungers 24, 28 to extend partially.
[0045] 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.
[0046] 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.
[0047] In some variations, the beads may be conjugated with an activating material such as an antibody such as immunoglobulin g.
[0048] 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.
[0049] Furthermore, depending upon the beads and the biological material to be concentrated, the number of back-and-forth cycles between the syringes 22, 26 can 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 channel 16 being 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 channel 16. Moreover, the method of cycling back-and-forth between the syringes 22, 26 facilitates an efficient mixing of beads with the blood plasma or PRP to produce a more uniform slurry with relatively less frothing.
[0050]
[0051] Although the withdrawal channel 40 is shown extending transversely relative to the fluid channel 38, the withdrawal channel 40 may be angled at a non-normal angle relative to the fluid channel 38 instead. Moreover, the assembly 30 may be used with conventional syringes while in other variations the ports 34, 36 may be keyed or modified to engage temporarily or permanently with specially configured syringes.
Example 1
[0052] In one example of a test which was performed, a first volume 50A of citrated bovine PRP was introduced directly into the first syringe 22 which was coupled to port 36 and the withdrawal channel 40 outlet was sealed via a cap, as shown in the perspective view of
[0053] The first plunger 24 was depressed forcing the first volume 50A of PRP to pass into the fluid channel 38 and into contact with beads 52 contained within. As the first volume 50A is further introduced, the PRP and beads may begin mixing as they are swept into the opposed second syringe 26 to capture a second volume 50B while forcing the second plunger 28 to expand, as shown in the perspective view of
[0054] The process of gently cycling the PRP back-and-forth between the syringes 22, 26 through the manifold body 32 may be maintained for a period of time, e.g., about 30 sec., to allow for the beads 52 to 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 syringe 54 may be attached to the withdrawal port 40 with its plunger fully depressed, as shown in the perspective view of
[0055] When the bulk of concentrated fluid 56 had been expressed through the filter or fit 42 into the collection syringe 54, the remaining bead pack 52 may appear uniform and white, indicating that the mixing and harvesting had maintained uniformity of the slurry and that concentrated fluid 56 had been almost entirely transferred out into the collection syringe 54, as shown by the perspective view of
Example 2
[0056] In another example of a test which was performed, a volume of buffy coat 60A comprised of plasma, platelets, white blood cells, and a small amount of red blood cells was introduced into the first syringe 22 which was coupled to port 36 opposite to the second syringe 26 which was coupled to port 34 with its plunger 28 fully depressed, as shown in the perspective view of
[0057] An amount of the beads 52 was preloaded into the first syringe 22 with the volume of buffy coat 60A although the beads 52 may be alternatively preloaded within the fluid channel 38 of the manifold body 32. The volume of buffy coat was then cycled between the syringes 22, 26 through the fluid channel 38 to thoroughly mix the beads 52 between the volumes 60A, 60B, as shown in the perspective view of
[0058] After a period of time, e.g., about 30 seconds, of cycling the mixed slurry back-and-forth between the syringes 22, 26, a collection syringe 62 was attached to the outlet port 40 and concentrated product 64 was expressed though the bead-retaining filter or frit 42 into the collection syringe 62 by continuing to pass the slurry back-and-forth between the in-line syringes 22, 26, as illustrated in the perspective view of
[0059] After concentrated product 64 was collected, the remaining bead pack 52 may appear uniform and slightly pink in color from traces of red blood cells in the small remaining volume of fluid interstitial between the packed beads 52.
[0060] 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 beads 52 employed. 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.
[0061] Alternative Configurations
[0062] 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 of
[0063] The biasing element 80 may maintain the plunger in a compressed state such that the plunger is biased to push away from the anchoring platform 80. With this configuration, the first syringe 22 may contain the beads 52, as shown, or the manifold body 70 may contain the beads 52. In either case, the blood plasma or PRP may be introduced into the first syringe 22 and the plunger 24 may be depressed to force the mixture of the beads 52 and blood plasma or PRP through the fluid channel of the manifold body 70 and into the second syringe 78. The plunger 24 of the first syringe 24 may be released to allow for the biasing element 80 to automatically push the plunger within the second syringe 78 to force the mixture back through the manifold body 70 and back into the first syringe 22. This process may be repeated, as described herein, until the fluid has been sufficiently concentrated. The stop cock 76 or valve (if present) may be opened to allow for the concentrated product to pass through the filter or frit 72 and through the withdrawal channel 74 and into the attached collection syringe 86.
[0064] In yet another embodiment as shown in the perspective view of
[0065] With the plunger of the first syringe 22 initially fully depressed, the plunger may be pulled proximally with the valve 91 closed such that a vacuum force is created within the first plunger 22. This also allows for the volume of beads 52 contained within to be dispersed within the syringe 22 prior to contacting the fluid contained within the second syringe 88. The first plunger may be locked into its retracted position via a locking mechanism 93 and the valve 91 may be opened so that the vacuum within the first syringe 22 may pull the fluid 89 in through the manifold 70 from the second syringe 88. This sudden dispersion of fluid 89 into the first syringe 22 may help to minimize any likelihood of clumping of the beads 52 within the first syringe 22 upon initial fluid contact and may also facilitate the rapid introduction of the fluid 89 after the valve 91 is opened.
[0066] Once the fluid 89 has been introduced into the first syringe 22, the locking mechanism 93 may be released (e.g., by twisting the plunger rod relative to the syringe body) and the cycling of the slurry between the two syringes 22, 88 may be accomplished. A collection syringe may be subsequently coupled to the manifold 70 for withdrawal, as previously described. Similarly, the syringes 22, 88 may be attached via a straight connector or a connector which adjoins the two syringes at any angle relative to each other.
[0067]
[0068] Because the ports 96, 98 are located along a common surface of the manifold body 92, the syringes may be angled relative to one another and relative to the manifold body 92 in any number of angles. Furthermore, this variation may be utilized with any of the devices and methods described herein.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] In yet another variation for recovering a volume of concentrated product, the coupling 110 shown in
[0073] With the mixed slurry introduced through port 114 and into the upper portion 120 of housing 112, both ports 114, 116 may be capped or closed and entire coupling 110 may undergo centrifugation to drive the fluid from the upper portion 120, through the fit 118, and into the lower portion 122 of housing 112. Once all concentrated product has been separated into the lower portion 122 and the beads remaining within the upper portion 120 above the fit 118, the withdrawal port 116 may be opened to withdraw the concentrated product from the housing 112.
[0074] In one example of use for the housing 120, a manifold 12 may be coupled to respective syringes 22,26 to 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 syringes 22,26, as described above and as shown in
[0075] Once the slurry or mixture 130 has been sufficiently transferred, the syringe 26 may be decoupled from the introduction port and housing 112 may then undergo centrifugation to drive the slurry or mixture 130 from the upper portion 120, through the frit 118, and into the lower portion 122 of housing 112. The frit 118 may retain the desiccating beads having absorbed one or more components from the blood plasma or PRP such that the resulting concentrated product 134 may be retained within the lower portion 122. An additional syringe 132 may be fluidly coupled to the withdrawal port 116 to then withdraw the concentrated product 134 from the lower portion 122 and into the additional syringe 132, as shown in
[0076] 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.