HK1190036B - Systems and methods for autologous biological therapeutics - Google Patents

Description

System and method for autologous biological therapy

Technical Field

Priority of the present application for U.S. patent application serial No.61/453,658 entitled "system and method for applying force to a tissue and tissue system," filed on day 3, month 17, 2011, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

Technical Field

The present invention relates to systems, methods and devices for autologous biological therapy, including but not limited to Platelet Rich Plasma (PRP) and Bone Marrow Cell Concentrate (BMCC) therapy and techniques.

Background

One of the validated methods to enhance hard and soft tissue regeneration is to add human growth factors to the wound site or surgical incision. A safe and simple way to obtain compatible growth factors in a clinical setting is to isolate platelets from the patient's blood, called "autologous platelet concentrate".

Platelets are blood cells that are primarily involved in hemostasis. However, they also contain proteins known as growth factors, which help promote healing and tissue regeneration. An artificial highly concentrated mixture of platelets (platelet concentrate or Platelet Rich Plasma (PRP)) has a higher platelet count than native blood, which has been found to stimulate the regeneration of soft and hard tissues of the body.

Bone Marrow Cell Concentrates (BMCCs) may include a number of cells of interest, including the following: stem cell-like cells (pluripotent cells) (e.g., monocytes), leukocytes, platelets, neutrophils, lymphocytes, eosinophils, and basophils, all of which have a variety of uses in healing, regeneration, and therapy. Since it is difficult to isolate any one or more of such cell fractions from the BMCC, they are typically all inserted or injected into the patient. In one embodiment, the BMCC includes platelets and leukocytes, including a stem cell fraction, wherein the stem cell fraction enhances the regenerative effect of the platelets.

Further understanding of the role of growth factors as biochemical regulators of wound healing has paved the way for a new family of bioactive therapeutic products to promote wound healing. Delivery of growth factors (recombinant or as autologous platelets) has emerged as a possible commercial opportunity for improving the clinical outcome of soft tissue, bone tissue and connective tissue repair. However, it is not possible in the art to control or manipulate the final product concentration within the narrow target range required to study or determine dose-response relationships and ultimately validate the therapeutic efficacy of such agents.

Although hematology analyzers (hematology machines) are capable of measuring typical platelet concentrations, they are not suitable for measuring the high platelet concentrations found in PRP transfusions. Such instruments are also very large and expensive (e.g., $15,000-.

Another challenge in forming platelet and BMC concentrates is that the separation and concentration process can prematurely activate platelets, thereby initiating the coagulation cascade. Therefore, there is a need for separation methods that avoid premature activation of platelets.

SUMMARY

Exemplary embodiments of the invention that are shown in the drawings are summarized below. These and other embodiments are described more fully in the detailed description section. It is to be understood, however, that there is no intention to limit the invention to the forms described in this summary of the invention or in the detailed description. One skilled in the art can recognize that there are numerous modifications, equivalents, and alternative constructions that fall within the spirit and scope of the invention as expressed in the claims.

In one aspect, the present disclosure describes a method of producing a platelet-rich plasma concentrate (or a bone marrow cell-rich plasma concentrate). The method may include separating a whole blood sample (or bone marrow sample) into a red blood cell fraction, a platelet-poor plasma (or bone marrow cell-poor plasma), and a platelet-rich plasma (or bone marrow cell-rich plasma). The method may further comprise determining the platelet concentration (or bone marrow cell concentration) by at least a first measurement before, during or after such isolation. The method may further comprise determining a first volume of fluid in which the first determination is made. The method may further include determining a second volume of platelet poor plasma (or bone marrow cell poor plasma) using the concentration and the first volume, the second volume of platelet poor plasma (component bone marrow cell plasma) when mixed with platelet rich plasma (or bone marrow cell rich plasma) will provide a concentration of platelets or bone marrow cells that falls within a target concentration range. Finally, the method may include producing a platelet-rich plasma concentrate (or bone marrow cell-rich plasma concentrate) by mixing a second volume of platelet-poor plasma (or bone marrow cell-poor plasma) with the platelet-rich plasma (or bone marrow cell-rich plasma).

In another aspect, the present disclosure describes a cell concentration system that includes a blood separation component, a measurement system, concentration and flow logic (concentration and flow logic), and a mixing component. The blood separation assembly may have a blood sample input and may be configured to separate the blood sample into an erythrocyte fraction and a plasma fraction. The plasma fraction may include a target cell-rich fraction and a target cell-poor fraction. The measurement system can measure the total number of target cells in the cell concentration system. The concentration and flow logic may determine a first volume of the target cell-poor fraction that is mixed with the target cell-rich fraction to form a target cell-rich concentrate. The concentrate can have a target cell concentration within a target concentration range. The mixing assembly can be configured to form a target cell-enriched concentrate by mixing a first volume of the target cell-depleted fraction with the target cell-enriched fraction.

In another aspect, the disclosure describes a method of producing a concentrate enriched in target cells. The method can separate a target blood sample into an erythrocyte fraction, a target cell-poor fraction, and a target cell-rich fraction. The method may also determine the total number of target cells in the target blood sample. In addition, the method can also calculate a first volume of the target cell-poor fraction such that when the fraction is mixed with the target cell-rich fraction, the combination will provide a target cell combination within a target concentration range. Finally, the method can include producing a target cell-enriched concentrate by mixing a first volume of the target cell-depleted fraction with the target cell-enriched fraction.

Brief description of the drawings

Various objects and advantageous aspects and a more complete understanding of the present invention will become apparent and more readily appreciated by reference to the following detailed description and appended claims when taken in conjunction with the accompanying drawings:

fig. 1 illustrates an operational and component flow diagram of a system for autologous infusion of platelet-rich plasma concentrate or bone marrow cell concentrate with specified concentration ranges.

Fig. 2 illustrates a method for producing a PRP concentrate with a specified platelet concentration.

Fig. 3 illustrates another method for producing a PRP concentrate with a specified platelet concentration.

Fig. 4 illustrates yet another method for producing a PRP concentrate with a specified platelet concentration.

FIG. 5 illustrates a method for producing a BMC-enriched concentrate having a specified BMC concentration.

Fig. 6 illustrates another method for producing a BMC concentrate having a specified BMC concentration.

Fig. 7 illustrates yet another method for producing a BMC concentrate having a specified BMC concentration.

Fig. 8 illustrates a block diagram representation of another system for generating a target concentration and/or volume of platelets or Bone Marrow Cells (BMCs).

FIG. 9 illustrates a PRP or BMC concentrator.

Fig. 10 illustrates one embodiment of a package containing a device component that may be disposable.

Fig. 11 illustrates how a syringe may be used to provide a whole blood or bone marrow sample to the device of fig. 9.

Fig. 12 illustrates a user interacting with the device of fig. 9 through, for example, a touch screen embodiment of a user interface.

Fig. 13 illustrates how a syringe may be used to remove the contents of the first container illustrated in fig. 9-13.

Fig. 14 shows a diagram of one embodiment of an apparatus in the exemplary form of a computer system in which a set of instructions are executable to cause an apparatus to perform or perform any one or more of the aspects and/or methods of the present disclosure.

Detailed Description

There is a long-felt need in the art for systems, methods, and devices that can produce PRP and BMCC in a narrow concentration range. With such known concentrations, clinical studies of the effect of different concentrations on patients would be greatly facilitated.

For purposes of this disclosure, Bone Marrow Cells (BMCs) and Bone Marrow Cell Concentrates (BMCCs) include, but are not limited to, any one or more of the following cells: stem cell-like cells (pluripotent cells) (e.g., monocytes), leukocytes, platelets, neutrophils, lymphocytes, eosinophils, and basophils. In fact, BMC includes any cell found in a bone marrow sample, and BMCC includes any cell found in a bone marrow sample (but at a concentration higher than the native concentration). Throughout this disclosure, a BMC-rich fraction refers to a substance or fluid having an increased concentration of any one or more target or desired cells as compared to the natural concentration in the human body. BMC-poor fractions refer to substances or fluids having a reduced concentration of any one or more target or desired cells compared to the natural concentration in the human body.

The concentrate of the present disclosure may include one or more of cells, signals, and scaffolds. Such components can be harvested and produced from a number of sources. For example, in an autologous system, a patient is treated with components taken from the same patient.

For purposes of this disclosure, "cells" include Mesenchymal Stem Cells (MSCs) derived from bone marrow, fat, blood, synovium, or other tissues. Cells also include pluripotent cells from bone marrow, blood, and other sources. Cells also include native tissue cells, which can be stimulated to grow and proliferate by signals.

For purposes of this disclosure, "signal" refers to a human growth factor protein, which may be derived from platelets or from an autocrine (cell-cell) source. Furthermore, for the purposes of this disclosure, "scaffold" refers to a mechanical matrix (mechanalmatrix) produced from blood-based fibrin that can be used to deliver and provide a platform for in vivo tissue growth. As disclosed herein, there is typically blood-based fibrin that creates a scaffold by inducing the coagulation cascade of blood, converts fibrinogen to fibrin, and creates a mechanical fibrin matrix. Cells and/or growth factor proteins may be implanted or 'seeded' into the scaffold, where the scaffold supports the formation of three-dimensional tissue from the seed.

Whole blood is human blood from standard blood donations. Whole blood may be combined with the anticoagulant during collection, but is typically not otherwise processed. By "whole blood" in case it is meant a specific standardized product for transfusion or further processing. Lower case "whole blood" (whole) includes any unmodified collected blood.

Flow Cytometry (FCM) is a technique for counting and examining microscopic particles, such as cells and chromosomes, by suspending them in a fluid stream and subsequently passing them through an electronic detection instrument. Which allows simultaneous multi-parameter analysis of physical and/or chemical characteristics of up to thousands of particles per second.

For the purposes of this disclosure, separation means chemically separating the components, but not necessarily physically separating. For example, centrifugation separates the fluid into layers that are separated from each other based primarily on the mass of the particles. However, there may be some overlap between the layers, and this is included in the application of the term separate in this disclosure. Also, if those layers are separated into different containers, this may also be considered to be a separation.

Fig. 1 illustrates an operational and component flow diagram of a system for autologous infusion of platelet-rich plasma concentrate or bone marrow cell concentrate with specified concentration ranges. Fig. 1 is described in conjunction with fig. 2-4, which describe the same method. A whole blood sample or Bone Marrow Cell (BMC) sample 102 is taken from a patient 104 (blocks 202, 302, 402, 502, 602, 702). The sample 102 enters a Red Blood Cell (RBC) separation assembly 106 (first separation assembly) that separates RBCs from a plasma fraction of the sample 102 (blocks 204, 304, 404, 504, 604, 704). RBCs may be stored in an RBC storage or processing container 108 (blocks 206, 306, 406, 506, 606, 706).

The plasma fraction may then be separated in a PRP or BMC fraction separation module 110 (second separation module). The second separation assembly 110 separates the plasma fraction into a Platelet-poor plasma (PPP) fraction and a Platelet-rich plasma (PRP) fraction, or a BMC-poor fraction and a BMC-rich fraction (blocks 210, 310, 410, 510, 610, 710). The PPP or BMC lean fraction may be stored in a PPP or BMC lean storage or treatment vessel 112.

The PRP or BMC-rich fraction may be mixed with an aliquot of the PPP or BMC-poor fraction in the mixing component 114 (blocks 212, 312, 412, 512, 612, 712). Alternatively, rather than removing the entire PPP or BMC-poor fraction and then remixing an aliquot of the removed fraction, a first aliquot of the PPP or BMC-poor fraction may be removed to the PPP or BMC-poor storage or processing vessel 112, while a second aliquot is left in the PRP or BMC-rich fraction.

The mixing assembly 114 may be controlled by a concentration and flow logic control 116. The concentration and flow logic control 116 determines the aliquot volume to be added to the PRP or BMC-rich fraction based on the determination of the total number of platelets or BMCs present in the whole blood sample or BMC sample 102 (blocks 208, 308, 408, 508, 608, 708). Such a determination may be made by measuring the platelet or BMC concentration 120,122,124 via the platelet or BMC concentration measurement component 118 at various points in the process. For example, the concentration 120 may be measured from a whole blood or BMC sample (blocks 408, 708). The concentration 122 may be measured after RBC separation, thereby measuring platelet or BMC concentrations in the plasma (blocks 208, 508). As another option, the concentration 124 may be measured after the PRP or BMC-rich fraction has been separated in the second separation assembly 110 (blocks 308, 608). This concentration 124 is measured from PRP or BMC-rich fractions. All 3 concentrations 120,122,124 should be the same measurement, although they may vary due to non-ideal separation (so that some platelets or BMC may end up in the RBC fraction or PPP or BMC depleted fraction). Since the blood sample and bone marrow cells may be separated, a stirring step may be added prior to measuring any of the first, second, or third concentrations 120,122, 124.

The total number of platelets or BMC in the PRP or BMC-rich fraction can be determined by multiplying the concentrations 120,122,124 by the volume of the sample 102, the plasma volume, or the volume of the PRP or BMC-rich fraction, respectively. Concentration and flow logic control 116 divides the total by the target concentration (e.g., 0.8-2.0 x 10)⁶Platelet/μ L or 1.0-1.5 x10⁶Platelets/. mu.L) to obtain a mixtureThe total target volume that the object should reach. The amount of PPP or BMC-poor fraction mixed with the PRP or BMC-rich fraction is the difference between the total target volume and the volume of the PRP or BMC-rich fraction (see equations 1-6 for further explanation).

An aliquot of PPP or an aliquot of the BMC-poor fraction and a mixture of PRP or BMC-rich fraction can be stored in a PRP or RMC-rich concentrate storage container 126. The mixture may also be referred to as a PRP or RMC-rich concentrate, and may have a target concentration and/or target volume of platelets or BMC. The PRP or RMC-enriched concentrate is then available for infusion back into the patient 104 (blocks 216, 316, 416, 516, 616, 716). PRP or BMC concentrates are compatible with blood that is cross-matched to the blood pool.

In an alternative, an optional anticoagulant 128 may be added to the whole blood sample or BMC sample 102 prior to the first separation to help prevent platelet activation (blocks 222, 322, 422, 522, 622, 722). Similarly, the solution 130 of the reversion anticoagulant 128 may be added to the PRP or BMC-enriched concentrate before or after the concentrate has reached the PRP or RMC-enriched concentrate storage container 126.

Another embodiment may optionally agitate the concentrate by the agitation assembly 132 before or after the concentrate reaches the PRP or BMC-enriched storage container 126. This agitation can help mix the concentrate and/or initiate activation of platelets in the concentrate. The agitation assembly 132 may be separate from or integrated with the PRP or RMC-enriched concentrate storage container 126. Agitation may include stirring in one embodiment.

User interface 134 may be used to interconnect with, control, and monitor the process through concentration and flow logic control 116. The user interface 134 may enable a user (e.g., a physician, nurse, or technician) to monitor parameters of the procedure and provide input such as a target concentration and/or target volume of PRP or RMC-enriched concentrate.

In one embodiment, another measurement and analysis of the PRP or BMC concentration of the concentrate is performed prior to infusion into the patient 104 (blocks 214,314,414,514,614, 714). If the concentrate falls within the target concentration range, the concentrate may be provided to the patient 104 (blocks 216, 316, 416, 516, 616, 716). However, if the concentration does not fall within the target range, the concentrate can be remixed with the RBC fraction (blocks 218, 318, 418, 518, 618, 718) to be re-passed through the process beginning with RBC separation (blocks 204, 304, 404, 504, 604, 704) until the PRP or RMC-rich concentrate falls within the target range. When determining the platelet or BMC concentration 120 from the sample 102 (blocks 408, 708), the total number of platelets (block 424) or BMC (block 724) may optionally be determined prior to the first separation (blocks 404, 704).

In one embodiment, an optional anticoagulant 128, such as ACDA, may be added to the whole blood or BMC sample 102 prior to the initiation of any separation process. Because the separation process and the mere movement of platelets between the container, centrifuge, or any other component of the system entails agitation of the platelets, and the agitation may initiate undesirable platelet activation (clotting), the anticoagulant 128 helps to hold the platelets in an inactive state until they are ready for infusion back into the patient 104. In one embodiment, 3mL of anticoagulant can be added to 50mL of whole blood or 5mL of anticoagulant can be added to 50mL of BMC. In the case of BMC sample 102, an aspirating syringe (aspiratonsyringe) may be flushed prior to passing BMC sample 120 through separation assembly 106. For example, the aspirating syringe can be flushed with heparin (e.g., 1,000U/mL).

In one embodiment, the whole blood or BMC sample 102 is 60-250mL, while in another embodiment, the sample 102 is 60-120 mL. Whole blood (wheelblood) may be obtained from an intravenous catheter. BMC may be collected by needle aspiration from the intramedullary cavity of the anterior or posterior hip, shoulder or knee, although other methods and sources for obtaining BMC are also contemplated.

While the presently discussed systems and methods describe autologous systems and methods (infusion back to the source patient 104), in other embodiments, the source patient and the patient to be infused may be different.

BMC sample 102 may be passed through an initial removal stage to remove high molecular weight components such as bone particles (bone particulate). The BMC sample 102 may then be filtered to remove any remaining fat and/or large particles (block 526,626,726). In one example, a 170-260 μm filter may be used.

A Red Blood Cell (RBC) separation assembly 106 separates the sample 102 into a RBC fraction and a non-RBC fraction or plasma fraction. In the case of the whole blood sample 102, the non-RBC fraction can include nucleated blood cells or White Blood Cells (WBCs), platelets, and serum. In the case of BMC sample 102, the non-RBC fraction can include plasma, platelets, and WBCs (including pluripotent cells).

The RBC separation assembly 106 can be ' soft spin ' (softspin) ' by centrifugation in one embodiment. In one embodiment, soft rotation may include centrifugation at 2500-. In centrifugation, the plasma fraction is the fraction of the sample 102 that accumulates above or closer to the center of the centrifuge (RBCs are the heaviest components of the RBC sample). RBCs accumulate below the plasma fraction as they tend to be heavier. Within the plasma fraction, centrifugation can also cause further separation between the primary plasma particles and the "buffy coat," which can include nucleated cells, platelets, plasma, and WBCs. The buffy coat tends to be present between the plasma and the RBC fraction.

The RBC separation assembly 106 can additionally employ a variety of other separation assemblies and methods, including but not limited to separation according to microfluidic channel separation methods, polymer-based separation, phonophoresis (acoustophoresis), various lab-on-chip or lab-on-CD (compact disc) technologies, flow cytometry, dielectrophoresis (dielectrophoresis), laser impedance (lasermpandance), flow cytometry, and the use of fluorescence or other labeling. Microfluidic channel separation methods involve passing a fluid through channels having different diameters so that particles having different sizes may fit through only certain channels, so that the particles may be separated depending on the channel through which they can pass. The acoustically induced ionic motion includes separating components of a fluid using an acoustic signal. The polymer-based method comprises adding a polymer to the plasma fraction that causes separation of platelets from plasma. The RBC separation module 106 should be selected to minimize platelet activation and maximize platelet yield.

The RBC fraction can be directed through a valve or pump into the RBC storage container 108 for later transfusion or processing. The plasma fraction may be directed to a different container or part of a system where further separation takes place. In some embodiments, the RBC fraction may remain in the first separation assembly 106 after the plasma fraction has been removed, and since the first separation assembly 106 may be disposable, the RBC fraction may remain in the first separation assembly 106 for processing. In such embodiments, a separate RBC storage or processing vessel 108 is not used.

In the case of a plasma fraction derived from bone marrow, a filtration process may be used to further filter the plasma (e.g., a 200 μm mesh filter). For example, any anticoagulant-flushed syringe may be used to push the plasma fraction through the filter.

Once the RBC fraction is separated from the plasma fraction, the plasma can be passed through a Platelet Rich Plasma (PRP) or BMC rich fraction separation module 110 (second separation module). The second separation assembly 110 separates the plasma into a Platelet Poor Plasma (PPP) fraction and a Platelet Rich Plasma (PRP) fraction or a BMC poor fraction and a BMC rich fraction. In many cases, the PPP fraction or BMC-poor fraction is the larger fraction. The PPP fraction tends to have a low or negligible concentration of platelets.

In some embodiments, the second separation assembly 110 is a RBC separation assembly 106 (also referred to as a first separation component), hereinafter referred to as separation assembly 106/110. For example, a single centrifuge may be used to separate sample 102 into an RBC fraction, a PRP or BMC-rich fraction, and a PPP or BMC-poor fraction. This may include first removing the plasma fraction from the separation module 106/110, then either returning the plasma fraction through the separation module 106/110 or leaving the plasma fraction in the separation module 106/110, while first separating and removing the RBC fraction, then separating the plasma fraction. Alternatively, the 3 fractions may be separated simultaneously.

In other embodiments, the first separation module 106 and the second separation module 110 are separate and distinct modules. For example, two centrifuges may be used. However, the first and second separation assemblies 106, 110 may also be different types of assemblies. In one instance, a centrifuge may be used as the first separation assembly 106 and a set of microfluidic pores having different diameters may be used as the second separation assembly 110. Many other variations are possible.

In case the second separation assembly 110 is a centrifuge, or in case both separation assemblies 106, 110 are the same centrifuge, a second or ' hard spinning ' (hardpin) ' of the plasma fraction may be performed. Hard rotation may include rotation at 2800-. The hard spin separates the plasma into a PPP fraction and a PRP fraction, or a BMC-poor fraction and a BMC-rich fraction, wherein the PPP fraction or BMC-poor fraction tends to be a larger upper layer containing lighter particles and only a small concentration of platelets or BMC. Below this layer is a smaller "buffy coat" or "pellet" containing heavier platelets or BMC, which accumulates toward the outer diameter of the centrifuge.

The PRP or BMC-rich fraction separation assembly 106 optionally uses a variety of other separation assemblies and methods, including but not limited to separation according to microfluidic channel separation methods, polymer-based separation, phonophoresis (acoustophoresis), various lab-on-a-chip or lab-on-CD (compact disc) techniques, flow cytometry, dielectrophoresis, laser impedance, flow cytometry, and the use of fluorescent or other labels.

Once the plasma fraction is separated, the PPP or BMC poor fraction may be directed to a PPP or BMC poor storage or processing vessel 112. For example, one or more valves and pumps may be used to direct fluid flow.

In one embodiment, rather than removing the PPP or BMC depleted fraction, only an aliquot of the fraction is removed to the storage or processing vessel 112. The selection of the volume of the aliquot will be discussed subsequently with respect to the concentration and flow logic control 116 and the mixing assembly 114.

The mixing assembly 114 may agitate the mixture of PRP and PPP or the mixture of BMC-rich and BMC-poor fractions to suspend the cells in the fluid. Alternatively, the effect of simply forcing the two substances into the same container can achieve satisfactory mixing.

The volume of an aliquot that was mixed with the RPR or BMC-rich plasma fraction to obtain the target concentration of PPP or BMC-poor plasma fraction was determined as follows. For the sake of readability, the description refers to platelets only, but is equally applicable to BMC. First, the total number of platelets P in the sample 102 is measured_{General assembly}（P_total). This can be done by: (a) measuring or estimating platelet concentration PC₀(e.g., platelet concentrations 120,122, 124) and (b) subjecting the platelet concentration PC to₀Multiplied by the volume V of the fluid in which the platelet concentration is measured₀. This is shown in the following equation (1):

(1)P_totat=PC₀XV₀

at least 3 measurable concentrations of PC are present in the process₀And volume V₀The location or time of. First, a first concentration 120 may be measured from the whole blood sample 102. Second, the second concentration 122 may be measured after the first separation assembly 106 has separated the sample 102 into an RBC fraction and a plasma fraction. The second concentration 122 may be measured before or after the RBC fraction is moved to the RBC storage or processing container 108. In summary, the second concentration 122 is obtained from the plasma fraction and not the RBC fraction. Third, the third concentration 124 may be measured after separating the plasma fraction into a RBC fraction and a PPP fraction.

The concentration of PC₀To the concentration and flow logic control 116 where it is used to determine the volume of fluid V being measured₀Total number of platelets in (a). The platelet or BMC concentration measurement component 118 can also measure the concentration PC therein₀Measured volume V₀(e.g., by a flow meter). Alternatively, it may be at a known volume V₀In space of (2) measuring concentration PC₀. For example, the sample 102 or plasma fraction or PRP can have a known volume. In another embodiment, a user (e.g., a doctor, nurse, or technician) may enter volume V0 into user interface 134, which provides volume V0 to concentration and flow logic control 116. Thus, concentration and flow logic control 116 may utilize the measured concentration PC of the fluid as per equation 1₀And volume V₀To calculate the total number of platelets P_{General assembly}（P_total）。

To obtain a target (e.g., user-determined) PRP concentration PC_tSome aliquot of the PPP fraction is mixed with the PRP fraction. Target PRP concentration PC_tIs in an exemplary range of 0.8-2.0 x10⁶Platelet/μ L or 1.0-1.5 x10⁶Individual platelets/. mu.L. An exemplary target PRP concentration PC_tIs 1.5x10⁶Individual platelets/. mu.L. In one embodiment, this comprises removing some of the PPP fraction, followed by mixing the remaining PPP fraction and PRP fraction. In another embodiment, the PPP fraction is removed and a portion of the PPP fraction is subsequently returned to mix with the PRP fraction. In both cases, it will be appreciated that the amount of PPP fraction mixed with the PRP fraction can be determined by calculating the target volume V of the mixture_TTo perform the measurement. The values are given as follows:

target volume V_tEqual to the number P of platelets_{General assembly}（P_total) Divided by the target PRP concentration PC_t. Equation (2) can be obtained by substituting equation (1) for P in equation (2) as follows_{General assembly}（P_total) To simplify:

to obtain the target PRP concentration PC_tThe PPP fraction can be divided into V_PPPAnd has a volume V_PRPThe PRP fractions (measured, for example, by a flow meter) are mixed so that the combination equals the target volume V_t. This can be written as equation (4), and the bisectors V in equations (5) and (6) are calculated as follows_PPP：

(4)V_tTwo V_PPP+V_PRP

Thus, in the case of removing a portion of the PPP fraction, some PPP fraction is removed until the volume of the remaining PPP fraction and PRP fraction is equal to V_t. In other words, a portion of the PPP fraction is removed until the volume of the remaining PPP fraction is equal to V in equation 6_PPP. After removal of the entire PPP fraction, will then have a volume V_PPPIn the case of addition of an aliquot of (b) back to the PRP fraction, the aliquot of the PPP fraction may be selected such that the volume V of the aliquot of the PPP fraction_PPPVolume V with PRP fraction_PRPIs equal to the target volume V_t。

Equation (6) may be influenced by the fact that some platelets are removed with the RBC by the first separation assembly 106 and with the PPP fraction by the second separation assembly 110; however, when the separation is carefully performed, such a number is negligible.

As noted earlier, the evolution of equation 6 is described for PRP, but is equally applicable to BMC-rich plasma. In particular, equation 7 shows equation 6 applied in the case where bone marrow is the source and BMC-rich plasma concentrate is the final target.

The concentration of BMC BMCC may be measured from BMC sample 102 after BMC sample 102 has been separated into a RBC fraction and a plasma fraction by first separation assembly 106, or after plasma fraction has been separated into a BMC-poor fraction and a BMC-rich fraction by second separation assembly 110₀. The target concentration of BMC was BMCC_t. The volume of the BMC-rich fraction was V_BMC+The volume of the aliquot of BMC poor fraction to be mixed with this BMC rich fraction is V_BMC. Again, an aliquot V of the BMC poor fraction can be prepared_BMCAdded to the BMC-rich fraction or left in the BMC-rich fraction (when the remaining fraction of the BMC-poor fraction is removed). Equations 6 and 7 may also apply to any target blood cells such as leukocytes (e.g., monocytes), platelets, bone marrow cells, and "stem cell-like" or "pluripotent" cells.

During this process, the concentration and flow logic control 116 may provide an indication to the user via the user interface 134 indicating the amount of PPP or BMC poor fraction mixed with the PRP or BMC rich fraction. Alternatively, the concentration and flow logic control 116 may provide the target volume V to the user via the user interface 134_tThe value of (c). In another embodiment, the concentration and flow logic control 116 may automatically control the mixing assembly 114 and control the volume of PPP or BMC-poor fraction mixed with the PRP or BMC-rich fraction. For example, the concentration and flow logic control 116 may control valves and pumps that take a certain amount of the PPP fraction or add a certain amount of the PPP fraction back to the PRP or BMC-rich fraction.

The user interface 134 may display information describing measurements made by the platelet or BMC concentration measurement component 118. The user interface 134 may be part of or separate from the concentration and flow logic control 116. This information may also be stored in memory or transferred to a central record keeping system by telemetry.

The user interface 134 may also be used by a user (e.g., a doctor, nurse, or technician) to set a target PRP or BMC concentration PC_tOr BMCC_tAnd a target volume V_t. The user interface 134 may also be used to request and display results from concentration analysis of PRP or BMC-enriched concentrates. Such analysis may be performed after the concentrate has been formed, but before the concentrate is administered to the patient to ensure that the desired concentration is obtained.

The platelet or BMC concentration measurement component 118 may include a variety of hematology analyzers and methods. For example, Fluorescence Activated Cell Sorting (FACS) can determine the concentration of specific pluripotent, stem-like cells in a fluid based on antibody cell surface markers. Other exemplary embodiments of concentration measurement components 118 include those used for optical microscopy, optical light scattering, and electrical impedance. Optical microscopy includes a computer-controlled pattern of counting and differentiating particles by shape and size, and shape recognition components and logic. In some cases, the process cannot be performed continuously, and thus it may be necessary to sample discrete fluid portions. In one embodiment, sampling of a thin fluid layer may be performed. Optical light scattering can use hydraulically focused fluid streams, which can count different types of cells and molecules, especially if fluorescent markers or antibodies are used. The electrical impedance may use a hydraulically focused fluid flow.

In some embodiments, any of these concentration measurement components 118 may be combined with a particle separation component. For example, microfluidic channel devices may be used to separate particles within the PRP fraction, while light scattering devices may be used to measure many particles in each fluid stream. The combination of a particle separation device with a particle counting device may be beneficial in cases where the particle counting device is unable to distinguish between different types of cells or particles within the same fluid stream.

In one embodiment, a blood analysis may be performed on the PRP or BMC-enriched concentrate (block 214,314,414,514,614,714). The blood analysis may be a further blood analysis in addition to or as an alternative to one or more previous blood analysis steps. For example, in one embodiment, a blood analysis may be performed on a whole blood sample or a BMC sample and on a concentrate. In another embodiment, the concentrate can be subjected to a blood analysis after the first separation.

In one embodiment, the autologous thrombin or fibrin matrix may be prepared from some or all of the PPP or BMC poor fraction in the PPP or BMC poor storage or treatment vessel 112 (block 220,320,420,520,620,720). The effect of the anticoagulant 128 may be reversed, for example, by adding CaCl (e.g., a 10% CaCl solution may be added to the PPP or BMC-poor fraction). The PPP or BMC-poor fraction and whatever substance is used to reverse the anticoagulant 128 may be agitated (e.g., for about 1 minute), resulting in the formation of a fibrin clot. Additional PPP or some BMC-poor fraction may be added to the mixture during or after clot formation. Further agitation may be performed and the mixture may be given time for the clot to continue to form. When clotting is complete, the clot can be removed and manually compressed (or recentrifuged for compression).

In the case of autologous thrombin formation, the prothrombin protein is cleaved during the reversal of the anticoagulant, thereby generating thrombin. Removal of the clot leaves serum and a low concentration of thrombin. Thrombin can be combined with implanted PRP to initiate and regulate the release of platelet growth factor, thereby stimulating a regenerative response.

A fibrin matrix or scaffold is formed when fibrinogen in the PPP is activated by calcium reversal of the anticoagulant or addition of autologous thrombin (to induce cleavage of fibrinogen into fibrin). The matrix or scaffold may be implanted to serve as a scaffold from which native or implanted cells may attach and proliferate to form new tissue.

The clot can be transferred to the implantation destination of the patient 104. The implantation destination may be a joint or region in which regeneration is desired or any other selected site of the patient 104. Upon removal of the clot, the remaining PPP or BMC poor can be used to promote platelet activation by combining with PRP or BMC rich concentrate in vitro (to produce PRP membrane) or in vivo (to produce activated PRP). Although thrombin or fibrin matrices may be administered autologous, they may also be implanted in another patient than patient 104.

The autologous thrombin or fibrin matrix may be removed by syringe and implanted into the patient 104. In one option, fibrin matrix or Autologous Platelet Gel (APG) can be generated autonomously from the activated PPP and a portion of the PRP concentrate. Other products include activated PPP containing autologous thrombin. Manual or autonomous operation may be performed in the operating room or at the desired treatment site.

PPP may comprise a cell-free blood fraction comprising-55% blood volume, 91% water content and residual proteins. Although PPP is preferably platelet-free, a small residual platelet fraction is actually observed.

In some embodiments, the platelet or BMC concentration measurement component 118 can also measure other blood component concentrations such as leukocyte concentrations. Other concentrations that may be measured include concentrations of erythrocytes, neutrophils, lymphocytes, monocytes, eosinophils and basophils. These alternative measurements may be used in embodiments where the concentration of non-platelet and non-BMC blood cells is also concentrated to a target concentration or target concentration range.

Sometimes the mixing of the mixing assembly 114 is insufficient to suspend the platelets in the PRP or BMC concentrate. In these cases, or to enhance platelet activation, the optional agitation assembly 132 may agitate the PRP or BMC concentrate. When BMC is used, the pellet can be reconstituted (increased fluid content) in one of 3 media (acellular bone marrow aspirate), plasma, or PRP) to facilitate infusion into the patient 104.

One or more of the first and second separation assemblies 108, 110, platelet or BMC concentration measurement assembly 118, concentration and flow logic control 116, and mixing assembly 114 may be discarded after use to enhance sterility within the system 300. All of the storage containers 108, 112, 126 may also be disposable.

The system 100 may take a variety of forms, including a miniaturized form such as a hand-held or desktop system or other portable instrument. Portable may include hand-held, lightweight, and/or vehicle-mounted support. The system 100 may also be designed to administer a continuous or intermittent infusion of PRP to a patient over a period of time. In one embodiment, system 100 may be operated, in whole or in part, as one or more micro-electro-mechanical systems (MEMS) devices and/or as a lab-on-a-chip.

The systems and methods disclosed herein may also achieve many other objectives through various alternative embodiments. In one embodiment, the sterility of the blood product is maintained, for example, by the inclusion of one or more disposable components. In one embodiment, blood is collected from a patient prior to surgery and used to prepare a final PRP product for surgery. In one embodiment, a PRP film is produced. Alternatively, activated PRP is used to produce PRP within an autologous fibrin matrix. In another embodiment, the system 100 may produce a PRP or BMC concentrate within 30 minutes.

The systems and methods may be integrated or integrated into existing technologies and components used in automated blood transfusion and blood analyzers. In addition to counting, measuring, and analyzing red blood cells, white blood cells, platelets, or other blood components, automated blood analyzers can also measure the amount of hemoglobin or chemical modulators in the blood and within each red blood cell.

The systems and methods described herein may be used for damaged or diseased tissue to stimulate and/or enhance repair or regeneration. Methods of implantation may include percutaneous (injection) or intra-operative (surgical) applications. Other embodiments may include implanted or partially implanted continuous or periodic delivery systems for providing a determined dose over time.

Other sample sources that may be used in place of whole blood and bone marrow include fat, synovium and other tissues.

Figure 2 illustrates a method for producing a PRP concentrate with a specified platelet concentration. The method 200 includes obtaining a whole blood sample in a obtain sample operation 202 and optionally adding anticoagulant in an add anticoagulant operation 222. The first separation operation 204 then separates the sample into a Red Blood Cell (RBC) fraction and a plasma fraction. The RBC fraction may be discarded or re-perfused into the patient in a discard or re-perfusion operation 206.

Total number of platelets P in plasma fraction_{General assembly}（_Ptotal) May be determined in a determining operation 208. This operation 208 utilizes at least one measurement, such as a concentration measured by a hematology analyzer. Can reduce the concentration of blood platelet PC₀Multiplied by the volume V of the liquid from which the concentration measurement is made₀(as measured, for example, by a flow meter). Volume V₀Multiplied by the concentration PC₀Obtaining the total number of platelets P_{General assembly}（P_total). The plasma fraction is then further separated by a second separation operation 210 (e.g., microfluidic channel separation or centrifugation) in which a Platelet Rich Plasma (PRP) fraction (high concentration of platelets) and a Platelet Poor Plasma (PPP) fraction (negligible platelet concentration) are produced.

Aliquot V of PRP fraction and PPP fraction_PPPMixed in a mixing operation 212. Volume V of aliquot_PPPCan be based on the target platelet concentration PC_tAnd total number of platelets P_{General assembly}(e.g., equation 6). The mixing operation 212 forms a PRP concentrate, which can then be provided to a patient in a PRP concentrate providing operation 216. The remaining PPP fraction may also be used to form an autologous thrombin preparation for implantation into a patient in the autologous thrombin preparation procedure 220.

Optionally, after the PRP concentrate is formed in the mixing operation 212, the platelet concentration can be checked to ensure that the platelet concentration falls within the target range (or within the margin of error of the target platelet concentration) in the optional decision 214. If decision 214 finds that the PRP concentrate falls within the target range or within the margin of error of the target concentration, the PRP concentrate may be provided to the patient. If not, the concentrate can be remixed with the RBC fraction and the process beginning with the first separation operation 204 can be repeated.

Since sedimentation may at least partially separate the concentrate into a PPP layer and a PRP layer, optionally an agitation operation may be used to suspend the platelets prior to infusion.

Fig. 3 illustrates another method for producing a PRP concentrate with a specified platelet concentration. The method 300 is almost identical to the method 200, except that the determining operation 308 is performed on the PRP fraction after the second separating operation 310, rather than between the first separating operation 304 and the second separating operation 310. In some embodiments, the first separation operation 304 and the second separation operation 310 may be performed in a single operation by a single separation component (e.g., a centrifuge that produces an RBC fraction, a PRP fraction, and a PPP fraction).

Fig. 4 illustrates yet another method for producing a PRP concentrate with a specified platelet concentration. Method 400 is nearly identical to methods 200 and 300, with the primary difference being that the whole blood sample is subjected to an assay operation 408 prior to either of the separation operations 404, 410.

Another difference between the methods 200 and 300 is that the total number of platelets in the whole blood sample based on the at least one measurement operation 408 may be performed in parallel with the optional operation 422 of adding anticoagulant to the whole blood sample. Alternatively, the two operations 408 and 422 may be performed at overlapping or non-overlapping times between the obtain whole blood sample operation 402 and the first separation operation 404.

The final difference is that after the optional red blood cell fraction is remixed 418 with the PRP concentrate, the method 400 may include an optional total platelet count determination 424 operation. This operation 424 may determine the total number of platelets in the mixture of RBCs and PRP concentrate after remixing the RBCs with PRP concentrate (if the PRP concentrate does not fall within the target concentration range according to decision 414). In some embodiments, the first separation operation 404 and the second separation operation 410 may be performed in a single operation by a single separation component (e.g., a centrifuge that produces an RBC fraction, a PRP fraction, and a PPP fraction).

FIG. 5 illustrates a method for producing an RMC-enriched concentrate having a specified BMC concentration. The method 500 includes obtaining a BMC sample in a obtain sample operation 502 and optionally adding anticoagulant in an add anticoagulant operation 522. The first separation operation 504 then separates the sample into a Red Blood Cell (RBC) fraction and a plasma fraction. The RBC fraction may be discarded or re-perfused into the patient in a discard or re-perfusion operation 506.

BMC total BMC in plasma fractions_{General assembly}（BMC_total) May be determined in a determining operation 508. This operation 508 utilizes at least one measurement, such as a concentration measured by a hematology analyzer. The BMC concentration can be adjusted₀Multiplied by the volume V of the liquid from which the concentration measurement is made₀(as measured, for example, by a flow meter). Volume V₀Multiplying by the concentration BMC₀Total number of BMCs BMC_{General assembly}. The plasma fraction is then further separated by a second separation operation 510 (e.g., microfluidic channel separation or centrifugation) in which a BMC-rich plasma fraction (high concentration of BMC) and a BMC-poor plasma fraction (negligible BMC concentration) are produced.

Aliquots V of BMC-rich and BMC-poor fractions_BMCMixing in a mixing operation 512. Volume V of aliquot_BMCBMCC based on target BMC concentration_tAnd total number of BMCs BMC_{General assembly}(e.g., equation 7). The mixing operation 512 forms a BMC concentrate, which may then be provided to the patient in an operation 516 of providing a BMC concentrate to the patient. The remaining BMC-poor fraction may also be used to form an autologous thrombin preparation for implantation into a patient in the autologous thrombin preparation operation 520.

Optionally, after the BMC concentrate is formed in the mixing operation 512, the BMC concentration may be checked to ensure that the BMC concentration falls within the target range (or within a margin of error of the target BMC concentration) in the optional determination 514. If the determination 514 finds that the BMC concentrate falls within the target range or within a margin of error of the target concentration, the BMC concentrate may be provided to the patient. If not, the concentrate can be remixed with the BMC fraction and the process can be repeated beginning with the first separating operation 504.

Since sedimentation may at least partially separate the concentrate into a BMC-poor layer and a BMC-rich layer, optionally a stirring operation may be used to suspend the BMC prior to infusion. The method may also include an optional filter plasma fraction operation 524 after the first separation operation 504. Optional filtering operation 526 can remove any remaining fat and/or large particles. 170-260 μm filters may be used in one example.

Fig. 6 illustrates another method for producing a BMC concentrate having a specified BMC concentration. The method 600 is almost identical to the method 500 except that the BMC fraction is measured 608 after the second separation operation 610 rather than between the first separation operation 604 and the second separation operation 610.

Fig. 7 illustrates yet another method for producing a BMC concentrate having a specified BMC concentration. Method 700 is nearly identical to methods 500 and 600, except for the major difference that measurement operation 708 is performed on a whole bone marrow sample prior to either of separation operations 704, 710.

Another difference between methods 500 and 600 is that operation 708 for determining a total number of BMCs in the bone marrow sample based on the at least one measurement may be performed in parallel with optional operation 722 for adding anticoagulant to the bone marrow sample. Alternatively, operations 708 and 722 may be performed at any overlapping or non-overlapping time between the obtain bone marrow sample operation 702 and the first separation operation 704.

The final difference is that after the optional operation 718 of remixing the red blood cell fraction with the BMC concentrate, the method 700 may include an optional BMC total determination operation 724. This operation 724 may determine the BMC total in the mixture of RBCs and BMC concentrate after remixing RBCs with BMC concentrate (if BMC concentrate does not fall within the target concentration range according to decision 714).

Fig. 8 illustrates a block diagram representative of another system 800 for producing a target concentration and/or volume of platelets or Bone Marrow Cells (BMCs). The system 800 obtains or is provided with a whole blood sample or BMC sample 802 from a patient 804. The sample may optionally be mixed with anticoagulant 806 prior to entering separator 808 (e.g., a microfluidic channel or centrifuge, to name two).

Separator 808 can separate sample 802 into two fractions: red Blood Cell (RBC) fraction and plasma fraction. Separator 808 can also separate sample 802 into 3 fractions: RBC fraction, target cell-poor fraction, and target cell-rich fraction. The target cells are the cells that are desired to be present in the final concentrate at a particular concentration. For example, platelets, leukocytes, bone marrow cells, pluripotent cells, and stem cell-like cells are some exemplary target cells. Any cell found in the bone marrow sample may be a target cell. The target cell-poor fraction is a fraction with negligible concentrations of target cells, while the target cell-rich fraction has greater than natural concentrations of target cells.

As each fraction exits the separator 808, the flow meter 810 can measure the volume of fluid exiting the separator 808. This data can be communicated to concentration and flow logic to control component 812. Concentration and flow logic control 812 controls a first controllable mixing assembly 818 to control the flow of fluid. The flow logic control 812 also determines the total number of target cells by multiplying the concentration of target cells exiting the separator 808 by the volume of fluid exiting the separator 808. Flow logic control 812 may also determine how and in what amount to mix the fluids to obtain a target concentration of target cells.

Data representative of flow rate, concentration, volume, and other parameters may be displayed to a user via user interface 814.

The concentration of the various particles and cells within each fraction is measured using a hematology analyzer 818. Hematology analyzer 818 can be embodied in a variety of devices and methods, such as Fluorescence Activated Cell Sorting (FACS), optical microscopy, optical light scattering, and electrical impedance, to name a few. The concentrations measured by the blood analyzer 816 are communicated to the concentration and flow logic control 812, which uses these measurements to determine the total number of target cells in the fluid. Based on the target cell population, the concentration and flow logic control 812 can determine instructions for the first controllable mixing component 818 (e.g., a pump or a valve or a combination of both).

The first controlled mixing assembly 818 directs the RBC fraction to a RBC storage or processing vessel 820. It also directs the lean target fraction to a lean target storage or treatment vessel 822. Finally, it directs the target-rich fraction to a target-rich storage vessel 824. The order in which the 3 fractions are introduced into their respective containers 820, 822, 824 is not limiting and it is contemplated that any combination or order may be possible.

The concentration and flow logic control 812 may also command a second controllable mixing component (e.g., a pump, a valve, or a combination of both) to add an aliquot of the lean target fraction to the entire target-rich fraction within the target-rich storage vessel 824. The volume of the aliquot is selected such that the combination within the target-enriched storage vessel 824 has target cells at a concentration or concentration range that meets the target concentration or target concentration range.

When this target concentration or concentration range is achieved, the target cell concentrate is present in the target-enriched storage vessel 824 and may be provided to the patient 804 (or another patient). The remaining depleted target fraction in the depleted target storage or processing vessel 822 may also be provided to the patient 804 (or another patient) in the form of an autologous thrombin or fibrin matrix.

Fig. 9 illustrates a PRP or BMC concentrator 900. The device 900 includes a disk centrifuge 902 for separating whole blood or BMC samples. The disk centrifuge 902 can separate the sample into a Red Blood Cell (RBC) fraction (outer layer), a Platelet Rich Plasma (PRP) or BMC rich fraction (middle layer), and a Platelet Poor Plasma (PPP) fraction or BMC poor fraction (inner layer). Whole blood or bone marrow samples may be provided to the centrifuge through opening 918, which may receive, for example, a needle of a syringe. The opening 918 may be configured to be in a closed condition unless a needle of a syringe is placed into the opening, thereby allowing fluid to pass into the disk centrifuge 902, but not escape through the opening.

The PPP or BMC depleted fraction may be first removed and may be passed through the flow meter 904 and the blood analyzer module 906 to the first container 910. The fluid may pass through the flow meter 904 or the blood analyzer module 906 in any order, although as illustrated, the fluid flow first passes through the flow meter 904. The flow meter 906 provides the volume of the PPP or BMC lean fraction. The withdrawal and flow of the PPP or BMC-lean fraction may be controlled by a computer-controlled valve/pump 908.

Once the PPP or BMC-poor fraction is removed from the disk centrifuge 902, the PRP or BMC-rich fraction becomes the lower or innermost layer and can then be removed. The PRP or BMC-rich fraction is passed through a flow meter 906, thereby providing a volume of the PRP or BMC-rich fraction to the logic (e.g., processor) of the device 900. The PRP or BMC-rich fraction can also be passed through a hematology analyzer module 906 that measures the total number of platelets or BMC in the PRP or BMC-rich fraction. Given this volume and total number of platelets or BMCs, the logic within the device may determine the concentration of platelets or BMCs within the PRP or BMC-rich fraction as the product of the total number of platelets or BMCs and the volume of the PRP or BMC-rich fraction. The PRP or BMC-rich fraction can be directed to a second container 912. The removal and flow of PRP or BMC-rich fractions can be controlled by computer controlled valve/pump 908.

The RBC fraction can be retained in a disk centrifuge, and since the centrifuge is disposable, no further action is required on the RBC fraction. Logic within the device 900 can determine that an aliquot of the PPP fraction or BMC-poor fraction of the target platelet or BMC concentration is to be mixed with the PRP or BMC-rich fraction. In one embodiment, the user may set the target concentration through the user interface 916 (see fig. 12). A second computer controlled valve/pump 914 may allow an aliquot to pass from the first container 910 to the second container 912 to form a PRP or BMC concentrate. An optional agitation machine (not illustrated) may be activated to enhance mixing of the PRP or BMC concentrate in the second container 912.

The remaining PPP fraction or BMC poor fraction and PRP or BMC concentrate can be removed from the containers 910, 912 through respective openings 920 and 922, which in one embodiment can be accessed through the needle of a syringe (see fig. 13). Also, the containers 910, 912 are separable and removable so that they may each be moved to a patient or storage facility for later use.

Although fig. 9 has been described in which fluid is taken from the disk centrifuge 902 in the middle of the centrifuge 902, in other embodiments, fluid may exit the disk centrifuge from other points. For example, the fluid may exit from the outer diameter of the centrifuge in some embodiments. Likewise, the order in which the different fractions are taken may vary. In some embodiments, printer 924 may provide a hard copy of the data from device 900.

To ensure sterility, those portions of device 900 that contact blood or bone marrow may be modular and disposable. Fig. 10 illustrates one embodiment of a package 1000 that contains those components of the device 900 that are disposable. Such components may include any one or more of the following illustrative components: centrifuge 902, centrifuge opening 918, hematology analyzer module 906, first computer controlled valve/pump 908, and first and second containers 910, 912. In addition, the disposable portion of the device 900 can include a fluid channel 924 between the centrifuge 902 and the computer controlled threshold/pump 908 and a fluid channel 926 connecting the first reservoir 910 with the second reservoir 912. Any 2 or more of these components may be interconnected to simplify and ease installation, removal, and shipping, and the interconnected packages 1000 may be replaced with similar or identical packages 1000.

FIG. 11 illustrates how a syringe 928 may be used to provide a whole blood or bone marrow sample to the device 900 of FIG. 9 by insertion through opening 918.

Fig. 12 illustrates a user interacting with the apparatus 900 of fig. 9, for example, through a touch screen embodiment of the user interface 916.

Fig. 13 illustrates how the contents of the first container 910 illustrated in fig. 9-13 can be removed using a syringe 930. As described, the first container 910 may contain a PPP fraction or a BMC-poor fraction, and a syringe may be used to aspirate some or all of the contents of the first container 910.

While fig. 9-13 have described the first container 910 as storing a PPP or BMC poor fraction generally and the second container 912 as storing a PRP or BMC rich fraction or a PRP or BMC concentrate, those skilled in the art will recognize that the two containers 910, 912 are interchangeable and thus are not limited to being to the right or left of the device 900.

The systems and methods described herein may be performed in an instrument, such as a computer system, in addition to the specific physical devices described herein. Fig. 14 shows a diagram of one embodiment of an apparatus in the exemplary form of a computer system 1400, in which a set of instructions may be executed to cause a device to perform or perform any one or more aspects and/or methods of the present disclosure. The components in fig. 14 are merely examples and do not limit the scope of use or functionality of any hardware, software, embedded logic components, or combination of two or more such components to perform a particular embodiment.

The computer system 1400 may include a processor 1401, a memory 1403, and a storage 1408, which communicate with each other and with other components via a bus 1440. The bus 1440 may also connect a display 1432, one or more input devices 1433 (which may include, for example, a keypad, keyboard, mouse, stylus, etc.), one or more output devices 1434, one or more storage devices 1435, and various physical storage media (physical storage media) 1436. All of these elements may be interconnected with the bus 1440 directly or through one or more interfaces or connectors (adaptors). For example, various physical storage media 1436 can be interconnected with the bus 1440 by a storage media interface 1426. Computer system 1400 may have any suitable physical form including, but not limited to, one or more Integrated Circuits (ICs), Printed Circuit Boards (PCBs), mobile hand-held devices (e.g., mobile phones or PDAs), laptop or notebook computers, distributed computer systems, computing grids (computing grids), or servers.

Processor 1401 (or Central Processing Unit (CPU)) optionally includes a cache memory unit (cachememory unit)1402 for temporarily storing instructions, data, or computer addresses locally. The processor 1401 is configured to facilitate execution of computer readable instructions. Computer system 1400 may provide functionality for processor 1401 that executes software embodied in one or more tangible computer-readable storage media, such as memory 1403, storage 1408, storage 1435, and/or storage medium 1436. The computer-readable medium may store software that performs certain embodiments, and the processor 1401 may execute the software. Memory 1403 can read the software from one or more other computer-readable media (e.g., mass storage 1435, 1436) or from one or more other sources via an appropriate interface, such as network interface 1420. The software may cause processor 1401 to perform one or more processes or one or more steps of one or more processes described or illustrated herein. Performing such processes or steps may include determining data structures stored in memory 1403 and modifying the data structures as instructed by the software.

Memory 1403 may include various components (e.g., machine-readable media), including, but not limited to, a random access memory component (e.g., RAM1404) (e.g., static RAM "SRAM," dynamic RAM "DRAM, etc.), a read-only component (e.g., ROM1405), and any combination thereof. ROM1405 may be used to transfer data and instructions uni-directionally to processor 1401 and RAM1404 may be used to transfer data and instructions bi-directionally to processor 1401. ROM1405 and RAM1404 may include any suitable tangible computer-readable media described below. In one example, a basic input/output system 1406(BIOS), containing the basic paths that help to transfer information between components within computer system 1400, such as during start-up (start-up), may be stored in memory 1403.

Fixed storage 1408 is bidirectionally coupled to processor 1401, optionally through storage control unit 1407. Fixed memory 1408 provides additional data storage capability and may also include any suitable tangible computer-readable media described herein. Memory 1408 may be used to store operating system 1409, EXEC1410 (executable), data 1411, API application software 1412 (application programs), and the like. Typically, although not always, the storage 1408 is a secondary storage medium (e.g., a hard disk) that is slower than primary storage (e.g., the memory 1403). The memory 1408 may also include an optical disk drive, solid-state memory (e.g., flash-based system), or any combination of the above. The information in storage 1408 may be integrated as virtual memory in memory 1403, where appropriate.

In one example, memory 1435 may be removably connected with computer system 1400 through memory interface 1425 (e.g., through an external port connector (not shown)). In particular, memory 1435 and associated machine-readable media may provide non-volatile and/or volatile storage of machine-readable instructions, data structures, program modules, and/or other data for computer system 1400. In one example, the software may reside, completely or partially, within machine-readable media on the memory 1435. In another example, software may reside, completely or partially, within the processor 1401.

Bus 1440 connects the multiple subsystems. In this context, a bus may include one or more data signal lines that serve a common function, as appropriate. The bus 1440 may be any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus (peripheralbus), a local bus (localbus), and any combination thereof (using any of a variety of bus architectures). By way of non-limiting example, such architectures include an Industry Standard Architecture (ISA) bus, an enhanced ISA (eisa) bus, a Micro Channel Architecture (MCA) bus, a video electronics standards association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCI-X) bus, an accelerated graphics port (accelerated AGP graphics port) bus, a hypertransport (htx) bus, a Serial Advanced Technology Attachment (SATA) bus, and any combination thereof.

The computer system 1400 may also include an input device 1433. In one example, a user of computer system 1400 can enter commands and/or other information into computer system 1400 through input device(s) 1433. Examples of input devices 1433 include, but are not limited to, alpha-numeric input devices (e.g., a keyboard), pointing devices (e.g., a mouse or touch pad), touch pads, joysticks (joysticks), game pads (gamepads), voice input devices (e.g., a microphone, an acoustic response system, etc.), optical scanners, video or still image capture devices (e.g., a camera), and any combination thereof. An input device 1433 can be connected to bus 1440 through any of a variety of input interfaces 1423 (e.g., input interface 1423), including, but not limited to, a serial port, a parallel port, a game port, a USB port, a FIREWIRE port, a THUNDERBOLT port, or any combination thereof.

In some embodiments, when computer system 1400 is connected to network 1430, computer system 1400 may communicate with other devices connected to network 1430, particularly mobile devices and enterprise systems (entrepresses systems). Communications with computer system 1400 may be transferred via network interface 1420. For example, network interface 1420 may receive incoming communications (e.g., requests or responses from other devices) in the form of one or more packets (e.g., Internet Protocol (IP) packets) from network 1430, and computer system 1400 may store the incoming communications in memory 1403 for processing. Computer system 1400 may similarly store outgoing communications (e.g., requests or responses to other devices) in one or more packets in inner layer 1403 and communicate from network interface 1420 to network 1430. The processor 1401 can access these communication packets stored in the memory 1403 for processing.

Examples of network interface 1420 include, but are not limited to, a network card, a modem, and combinations thereof. Examples of network 1430 or network segment (network) 1430 include, but are not limited to, a Wide Area Network (WAN) (e.g., the internet, an enterprise network), a Local Area Network (LAN) (e.g., a network associated with an office, building, campus, or other relatively small geographic space), a telephone network, a direct connection between two computing devices, and any combination thereof. Networks such as network 1430 may utilize wired and/or wireless communication modes. In general, any network topology (network topology) may be used.

Information and data can be displayed via display 1432. Examples of the display 1432 include, but are not limited to, a Liquid Crystal Display (LCD), an organic liquid crystal display (OLED), a Cathode Ray Tube (CRT), a plasma display, and combinations thereof. A display 1432 may be connected to the processor 1401, memory 1403 and fixed storage 1408, as well as other devices such as an input device 1433, by a bus 1440. The display 1432 is connected to the bus 1440 through a video interface 1422, and the transmission of data between the display 1432 and the bus 1440 can be controlled by a graphics controller 1421.

In addition to the display 1432, computer system 1400 may include one or more other peripheral output devices 1434, including, but not limited to, speakers (audiopeak), printers, and combinations thereof. Such peripheral output devices may be connected to the bus 1440 through output interface 1424. Examples of output interface 1424 include, but are not limited to, a serial port, a parallel, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combination thereof.

Additionally or alternatively, the computer system 1400 may provide functionality as a result of logic, either hardwired or otherwise embodied in circuitry, that may operate in place of or in conjunction with software to perform one or more processes or one or more steps of one or more processes described or illustrated herein. The software referred to in this disclosure may comprise logic, which may comprise software. Further, a computer-readable medium may include circuitry for execution (e.g., an IC) to store software, circuitry for execution to embody logic, or both, as appropriate. The present disclosure includes any suitable combination of hardware, software, or both.

Those of skill in the art would understand that information and signals may be presented using any of a variety of different technologies and techniques. For example, data, instructions, commands, information, signals, bits, symbols, and integrated circuits that may be referenced throughout the above description may be represented by voltages, currents, electromagnetic waves, magnetic fields or particles, optical fields or particles, or any combination thereof.

Those of skill would further appreciate that the various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. Whether such functionality is implemented as hardware or software depends upon the particular application and design constraints imposed on the overall system. Skilled artisans may implement the described functionality in varying ways for each particular application, but such implementation decisions should not be interpreted as causing a departure from the scope of the present invention.

The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with a general purpose processor, a Digital Signal Processor (DSP), an Application Specific Integrated Circuit (ASIC), a Field Programmable Gate Array (FPGA) or other programmable logic device, discrete gate or transistor logic, discrete hardware components (discrete hardware components), or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.

The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by a processor, or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium known in the art. An exemplary storage medium is coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal.

In summary, the present invention provides, among other things, systems and methods for autonomously or semi-autonomously producing a PRP or BMC concentrate having a target concentration of platelets or BMC. One skilled in the art can readily recognize that numerous variations and permutations of the present invention, its uses and its configuration are possible with substantially the same results as achieved by the embodiments described herein. For example, blood products may be moved through the systems 100 and 800 or to other systems, either manually or autonomously. As another example, methods for separating blood components (e.g., microfluidic channel separation or electrical impedance separation) may be used in addition to centrifugation. Therefore, it is not intended to limit the invention to the exemplary forms disclosed. Many variations, modifications and alternative constructions fall within the scope and spirit of the disclosed invention.

Claims (21)

1. A cell concentration system, comprising:

a blood separation assembly having a blood sample input and configured to separate the blood sample into an erythrocyte fraction and a plasma fraction, wherein the plasma fraction comprises a target cell-rich fraction and a target cell-poor fraction;

a measurement system that measures a total number of target cells in the cell concentration system;

determining a concentration logic and a flow logic for a first volume of a target cell depleted fraction that is mixed with the target cell enriched fraction to form a target cell enriched concentrate, wherein the target cell concentration in the target cell enriched concentrate is within a target concentration range for studying or determining a dose-response relationship in a patient;

decision logic that determines whether a concentration of target cells in the target cell-enriched concentrate is within the target concentration range; and

a mixing assembly configured to form the target cell-enriched concentrate by mixing the first volume of the target cell-depleted fraction with a target cell-enriched fraction;

wherein the target concentration range is 0.8-2.0 × 10⁶Target cells/. mu.l.

2. The cell concentration system of claim 1, wherein the blood sample is a whole blood sample or a bone marrow sample.

3. The cell concentration system of claim 1, wherein the measurement system comprises:

a concentration measurement component that measures a concentration of a target cell in the cell concentration system; and

a flow meter assembly that measures a second volume in which concentration measurements are made.

4. The cell concentration system of claim 3, wherein the concentration measurement component measures a concentration of target cells in the target cell-enriched fraction and the second volume is a volume of the target cell-enriched fraction.

5. The cell concentration system of claim 4, wherein the total number of target cells is equal to the concentration of target cells multiplied by the second volume.

6. The cell concentration system of claim 3, wherein the concentration measurement component performs a function selected from the group consisting of: optical microscopy, optical light scattering and electrical impedance.

7. The cell concentration system of claim 1, wherein the blood separation module performs a function selected from the group consisting of: centrifugation, laser impedance, flow cytometry, dielectrophoresis, microfluidic channel separation, electrical impedance, and the use of fluorescent markers.

8. The cell concentration system of claim 1, wherein the mixing component is a valve or a pump.

9. The cell concentration system of claim 1, wherein the target cell is selected from the group consisting of: pluripotent cells, leukocytes, erythrocytes, platelets, neutrophils, monocytes, lymphocytes, eosinophils, and basophils.

10. A method of producing a platelet rich plasma concentrate or a bone marrow cell rich plasma concentrate using the cell concentration system of any one of claims 1-9, comprising:

separating the whole blood sample or bone marrow sample into a red blood cell fraction, platelet-poor plasma or bone marrow cell-poor plasma, and platelet-rich plasma or bone marrow cell-rich plasma;

determining a platelet concentration or a bone marrow cell concentration by at least a first measurement before, during or after the isolating;

determining a first volume of fluid in which a first measurement is made;

using said concentration and said first volume to determine a second volume of platelet poor plasma or bone marrow cell poor plasma that, when mixed with said platelet rich plasma or bone marrow cell rich plasma, will provide a platelet or bone marrow cell concentration that falls within a target concentration range for studying or determining a dose-response relationship in a patient;

determining whether the concentration of platelets or bone marrow cells in the platelet rich plasma or bone marrow cell rich plasma is within the target concentration range; and

producing a platelet-rich plasma concentrate or a bone marrow cell-rich plasma concentrate by mixing the second volume of platelet-poor plasma or bone marrow cell-poor plasma with the platelet-rich plasma or bone marrow cell-rich plasma;

wherein the target concentration range is 0.8-2.0 × 10⁶Target cells/. mu.l.

11. The method of claim 10, wherein the separating comprises:

separating the red blood cells from the whole blood sample to form a plasma fraction; and

separating the plasma fraction into a platelet poor fraction and a platelet rich fraction.

12. The method of claim 11, wherein the separating comprises:

centrifuging a whole blood sample to form a plasma fraction towards an inner diameter of a centrifuge and a red blood cell fraction towards an outer diameter of the centrifuge;

removing the red blood cell fraction;

centrifuging the plasma fraction to form a platelet poor plasma fraction towards an inner diameter of the centrifuge and a platelet rich plasma fraction towards an outer diameter of the centrifuge.

13. The method of claim 12, wherein the measurement is performed on a whole blood sample or a bone marrow sample.

14. The method of claim 12, wherein said measuring is performed on said plasma fraction after a first centrifugation.

15. The method of claim 12, wherein said measuring is performed on said platelet rich plasma fraction or said bone marrow cell rich plasma fraction after a second centrifugation.

16. A method of producing a target cell-enriched concentrate using the cell concentration system of any one of claims 1-9, comprising:

separating the target blood sample into an erythrocyte fraction, a target cell-poor fraction and a target cell-rich fraction;

determining the total number of target cells in the target blood sample;

calculating a first volume of a target cell-poor fraction that when mixed with the target cell-rich fraction will provide a target cell concentration within a target concentration range; and

producing a target cell-enriched concentrate by mixing a first volume of the target cell-poor fraction with the target cell-enriched fraction.

17. The method of producing a target cell-enriched concentrate of claim 16, wherein the target blood cells are selected from the group consisting of: pluripotent cells, leukocytes, erythrocytes, platelets, neutrophils, monocytes, lymphocytes, eosinophils, and basophils.

18. The method of producing a target cell-enriched concentrate according to claim 16, wherein the target blood sample is a whole blood sample or a bone marrow sample.

19. The method of producing a target cell-enriched concentrate of claim 16, wherein the determining comprises:

determining a target cell concentration by at least a first measurement;

determining a second volume of fluid in which a concentration of the target cells is determined; and

multiplying the target cell concentration by a second volume of the fluid to obtain a total number of target cells.

20. The method of producing a target cell-enriched concentrate according to claim 19, wherein the target cell concentration is determined before or after said isolating.

21. The method of producing a target cell-enriched concentrate of claim 19, wherein the separating comprises a first and a second separating, and wherein the determining of the target cell concentration is performed between the first and the second separating.