Method of preparation of mononuclear-platelet matrix (MPM) and related apparatus

Abstract

A method and related apparatus for separating plasma, lymphocytes, monocytes, and platelets from the denser phases of a sample of whole blood or pretreated cell fraction thereof in which a container is provided having an open end and a closed end. The container includes a first layer of a density gradient fluid at a first position in the container, a foam barrier contained at a second position, and an anticoagulant solution being located at a third position within the container in closer proximity to the open end of the container than the second layer. The anticoagulant solution has an effective concentration of sodium citrate with a pH adjusted to modify the pH of the anticoagulant solution, sufficient for preventing coagulation when the sample or pretreated cell fraction is added to the container, the anticoagulant solution having an adjusted pH ranging from pH 6.5 to pH 8.5.

Description

METHOD OF PREPARATION OF MONONUCLEAR-PLATELET MATRIX (MPM) AND RELATED APPARATUSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority based on applicable portions of 35 U.S.C §119 and 35 U.S.C. §120 to U.S. Patent Application Serial No. 63 / 635,156, filed April 17, 2024, the entire contents of which is herein incorporated by referenceTECHNICAL FIELD

[0002] The present invention generally relates to blood cell processing techniques and related apparatus. More particularly, the present invention relates to methods for separating platelets and mononuclear cells, such as lymphocytes and monocytes, together from whole blood specimens, especially assemblies or methods which maintain blood in an anticoagulated state, prior to partitioning the sample into discrete layers using centrifugation. Additionally, the present invention relates to blood separation assemblies or devices that provide a high recovery of platelets and mononuclear cells without significant contamination by red blood cells or polymorphonuclear granulocytes, including neutrophils.BACKGROUND

[0003] Acute wounds follow an organized wound healing sequence and often heal between 3 and 4 weeks. When a wound is still present 4 weeks after wounding, it is defined as a chronic wound. Many research studies have been conducted on chronic wound management to address the rising demand for effective and affordable care. The healing trajectory of a chronic wound is expected to take 12 weeks. This period may be prolonged if the wound presents with an altered molecular environment, chronic inflammation, fibrosis, or uncorrected preexisting systemic factors.

[0004] Chronic wounds, the most common of which are diabetic foot ulcers (DFU), pressure ulcers (PU), venous leg ulcers (VLU), and nonhealing surgical wounds, are a major healthcare problem. Chronic wounds usually occur in older individuals with underlying conditions such as diabetes mellitus, vascular disease, and obesity . Compromised immune andnutritional status, as well as chronic mechanical stress, have also been shown to contribute to poor wound healing outcomes. Chronic wounds are associated with alarmingly high mortality: the 5-year mortality rates of ischemic (55% mortality rate), neuropathic (45%), and neuroischemic (18%) diabetic foot ulcers, are higher than or similar to those associated with breast cancer and prostate cancer (18% and 8%, respectively). Chronic wounds are also associated with high healthcare costs: in the USA, in which total spending estimates for chronic nonhealing wounds have ranged from US$28. IB to US$96.8B in 2014, according to a retrospective analysis of the Medicare 5% Limited Data Set . Despite the alarming prevalence and high costs of care, efficient treatments are still lacking.

[0005] The complexity and multiplicity of the diabetic foot wound, for example, make it an immensely challenging therapeutic target, and the lopsided progress seen in murine models highlights the need for new methods to overcome the bench-to-bedside barrier. Clinical progress requires more innovative research strategies that harness both the existing knowledge and the potential of new advances across disciplines.

[0006] Blood platelets are small bioactive anuclear cells with diameters that vary between 2 and 4 pm and are derived from mature megakaryocytes in the bone marrow and lungs. Platelets are essential for primary hemostasis, but they also play important roles in tissue regeneration and inflammation. There are clear sex-and age-dependent differences in platelet characteristics, including count, mean volume, biological properties (e.g., mRNA status, oxidative stress, amino acid intake), oxygen metabolism, aggregation, and membrane fluidity. Platelet count remains relatively stable during middle age (25-60 years), but decreases in elderly people.

[0007] Platelet a-granules constitute the major granule population in terms of size and number within a platelet. These granules contain adhesion and growth factors, such as transforming growth factor-0 (TGF-0), platelet-derived growth factor (PDGF), platelet-derived endothelial cell growth factor (ECGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF), aswell as P-selectin, platelet factor 4, fibronectin, Beta-thromboglobulin, von Willebrand Factor (vWF), fibrinogen, and coagulation factors V and XIII.

[0008] Blood platelets are critical for hemostasis and thrombosis and play diverse roles during wound and immune responses, mainly consisting in rolling, adhesion, and aggregate formation in their local microenvironment. Platelets migrate to sites of vascular injury and inflammation and pile up on the adhesive substrate together with any bound particulate material. The platelets use this ability to act as cellular scavengers, scanning the vascular surface for potential invaders, and collecting deposited bacteria. Microbe collection by migrating platelets boosts the activity of professional phagocytes, such as monocytes, and assigns platelets a central role in innate immune responses.

[0009] Platelets have been used in wound care for several decades. The benefits of platelet rich plasma (PRP) administration are associated with an economical advantage, taking into consideration that PRP administration does not require complex equipment or training for its execution. Moreover, due to their primary autologous origin, concerns of disease transmission or immunogenic reactions can be disregarded. Platelets are easily available in large quantities from blood. A normal platelet count ranges from 150,000 to 450,000 platelets per microliter of blood. A platelet has 50-80 alpha granules which release hundreds of bioactive proteins including growth factors, adhesion molecules, and serotonin, which promotes cellular viability, proliferation, and migration. Platelet-derived microparticles (PDMs) stimulate the release of cytokines, activate intracellular signaling pathways, promote angiogenesis, and are involved in tissue regeneration. Platelets interact with immune cells and have analgesic effects. Platelets are involved in tissue remodeling and recruit bone marrow derived progenitor cells and mesenchymal stem cells. Platelets are also important for the maintenance of vascular integrity. Platelet mediators stimulate extracellular matrix formation and connective tissue restructuring.

[0010] For the treatment of chronic non-healing cutaneous wounds, technology has been developed to insert concentrated platelets into the wound to speed the wound healing process. Platelets isolated from the peripheral blood are an autologous source of growthfactors. The term "platelet-rich plasma" (PRP) was introduced in the 1970s to describe the autologous preparations and enrichment of platelets into a plasma concentrate. PRP, also known as autologous conditioned plasma, is a concentrate of platelet-rich plasma derived from whole blood, which has been centrifuged to remove red and white blood cells. Autologous PRP gel consists of platelets releasing cytokines, growth factors, chemokines, within a fibrin scaffold derived from a patient's blood. The mechanism of action for PRP gel is thought to be the molecular and cellular induction of normal wound healing responses similar to that seen with platelet activation. When PRP is activated, platelets release their growth factors, such as PDGF, fibroblast growth factor (FGF), TGF-0, EGF, and VEGF. These growth factors are involved in all stages of wound healing. Improved wound healing qualities have been attributed to PRP by the multitude of growth factors delivered to a wound.

[0011] For example, platelet-derived TGF- does not directly enhance the rate of wound closure but does appear to significantly promote keratinocyte proliferation and epidermal layer remodeling and regeneration. Use of PRP gel resulted in improved quality of life and lower cost of care over a 5-year period than other treatment modalities for nonhealing diabetic foot ulcers. Although actual treatment outcomes may differ from those modeled, PRP gel represents a potentially attractive treatment alternative for insurers and health care providers to address the cost burden and health effects of nonhealing diabetic foot ulcers.

[0012] Considerable research has been conducted in recent years to develop improved methods and devices for separating platelets from whole blood. Effective separation and isolation of platelets is often critical to various to therapeutic protocols. Consequently, a variety of blood collection / separation devices have been developed. For example, Nuo Therapeutics, Inc., has developed the Aurix System PRP gel; Zimmer-Biomet has developed the GPS® III Platelet Concentration System; Regen Lab has developed the Autologous Platelet Rich Plasma gel separator tube; APEX Biologix has developed the XCELL PRP™; AcCELLerated Biologies has developed the PurePRP® SP-SupraPhysiologic; Estar Medical has developed the Tropocells® Platelet Rich Plasma system; Reapplix A / S has developed the 3C Patch®; Arthrex has developed the ACP® PRP double-syringe system; Neogenesis Co., Ltd., has developed the GENESIS PRP;YCELLBIO MEDICAL Co. Ltd., has developed the PRP KIT; MacroCure has developed theCureXcell™; BTI Biotechnology Institute has developed the PRGF®-Endoret®; Curasan has developed the PRP Kit; PLATELTEX has developed the Plateltex-Act®; Vivostat has developed the Vivostat PRE; Arteriocyte Medical Systems has developed the Magellan® PRP™; AVITA Medical has developed the RECELL System; and PALL Corporation has developed the V-PET. Each of the above-listed systems prepare a platelet rich plasma, a platelet poor plasma, a platelet-rich fibrin matrix, or a leucocyte-platelet rich plasma. Some of the above-listed systems use thrombin or batroxobin to activate the platelets.

[0013] However, none of the systems prepare a concentrated mononuclear (monocytes and lymphocytes) cell preparation with platelets in plasma using a phase separator device without the use of thrombin or batroxobin. In accordance with the present invention, the advantage of combining mononuclear cells with platelets for wound healing combines the immune function of the mononuclear cells with the cell signaling of the platelets. Granulocytes are excluded since they contribute to inflammation. The use of PRP rich in neutrophils could result in a higher collagen type III to collagen type I ratio, adding to fibrosis and decreased tendon strength. Other neutrophil-mediated deleterious properties include the release of inflammatory cytokines and matrix metalloproteinases (MMPs) that promote pro-inflammatory and catabolic effects when applied to tissues. Neutrophils can produce extracellular traps (NETs), large extracellular web-like structures composed of decondensed chromatin bound to various cytosolic and granule proteins. While originally recognized as a defense mechanism against pathogens, neutrophils can hinder regeneration. In addition to the presence of neutrophils shortly after wounding, neutrophils remain within the wound after the NET barrier is reestablished. Taken together, study results demonstrate that although neutrophils are stimulated by a common pro-regenerative cue, their presence and NET formation can hinder regeneration.

[0014] Monocytes typically circulate through the blood for 1 to 3 days before migrating into tissues, where they become macrophages or dendritic cells. Macrophages exhibit plasticity and adopt pro-inflammatory, pro-wound-healing, pro-fibrotic, anti-inflammatory, anti-fibrotic, or tissue-regenerating phenotypes. According to their activation state and functions, macrophages can be divided into Ml-type (classically activated macrophage) and M2-type(alternatively activated macrophage). The balance between Ml and M2 macrophages plays an important role in wound healing. Most well characterized, the Ml macrophage initiates the pro-inflammatory immune responses to pathogen colonization. Recent advances describing Ml polarization suggest activation of circulating macrophages to this phenotype results in development of systemic inflammatory response syndrome in humans, demonstrating the lethality of inappropriate Ml sustained activation. As the inflammation abates, these alarmins are believed to facilitate the onset of anti-inflammatory or wound healing responses characterized by the presence of alternatively activated macrophages, or the M2 phenotypes.

[0015] Interferon gamma (IFN-y) can differentiate macrophages into Ml macrophages that promote inflammation. Macrophages are involved in the elimination of pathogens in tissues. When activated, macrophages can engulf and kill pathogenic microorganisms, release pro-inflammatory factors, and collect and activate lymphocytes to induce an adaptive immune response. Interleukin 4 (IL-4) produced by T helper type 2 (Th2) cells can convert macrophages into M2-type macrophages that inhibit inflammation. M2 macrophages mainly secrete antiinflammatory cytokines, which have the function of reducing inflammation and play an important role in wound healing and tissue repair.

[0016] Macrophage functional diversity in phenotype has recently evolved from the duality of Ml and M2 to include an additional four (4) alternately activated sub-phenotypes (M2a, M2b, M2c and M2d). Metabolic pathway utilization shifts characterize macrophage polarization with resulting metabolic and immune outcomes impacting host-pathogen interactions during wound healing. Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to the proliferative phase of healing. Macrophages isolated from wounds of patients afflicted with diabetes and from healthy controls have been found to have differential expression of the SET [Su(var)3-9, Enhancer-of-zeste and Trithorax] Domain Bifurcated Histone Lysine Methyltransferase 2 (Setdb2). The SET domain is a protein domain that typically has methyltransferase activity. For example, the Setdb2 encodes a member of a family of proteins that contain a methyl-CpG-binding domain (MBD) and a SET domain and function as histone methyltransferases. This protein is recruited to heterochromatin and plays a role in the regulation of chromosome segregation. Setdb2regulates macrophage plasticity during normal and pathologic wound repair. Setdb2 is increased in normal wound macrophages and may initiate the transition of wound macrophages from an inflammatory to a reparative phenotype during normal wound healing. Increased expression of Setdb2 serves as a brake on inflammation in which the inflammatory transcriptional program gets turned off and macrophages switch to a reparative phenotype.

[0017] Studies in mice have shown that a specific combination of growth factors (GFs) enhances the survival, adhesion, and angiogenic potential of mononuclear cells. In vivo wound healing results revealed that GF-treated wounds demonstrated accelerated wound healing at days 7 and 14, compared with those that are untreated. These histological analyses demonstrate that the number of engrafted cells and transdifferentiated keratinocytes in the wounds were significantly higher in the GF-treated subjects. This suggests that priming of mononuclear cells with growth factors released by platelets can enhance cell-based therapies.

[0018] Macrophages enter a wound and produce IL-10, which can then cause the cells that are around the wound to start closing the wound. Studies of mucosal wounds defined some of the signaling pathways that IL-10 uses to orchestrate wound repair. It was found IL-10 promotes intestinal epithelial wound repair through the activation of cAMP response elementbinding protein (CREB) signaling at the sites of injury, followed by synthesis and secretion of the WNTl-inducible signaling protein 1 (WISP-1). IL-10 also suppresses immune cell pro- inflammatory responses and plays an important role in maintaining homeostasis. This data highlights an important macrophage-epithelium crosstalk that is mediated by IL-10, CREB, and WISP-1 and that serves to promote healing of mucosal wounds.

[0019] Lymphocytes have a role in regulating the direction of wound repair, with or without scaring. T lymphocyte subsets, particularly Regulatory T cells (Tregs), have been shown in mice to attenuate the degree of inflammation and promote relevant neovascularization, thereby reducing the risk of dermal scarring. The time course of T lymphocyte infiltration into the wound shows that CD3+ T lymphocytes are present in the wound at day 3, peak at day 14, and persist until day 30, suggesting a significant T lymphocyte role in dermal wound healing and scarring responses. In the B and T lymphocyte-deficient SCID mouse, wound repair results inaccelerated epithelial wound closure, increased inflammation, decreased angiogenesis, and exacerbated scar formation. CD4+ T lymphocytes may represent the key lymphocyte population that regulates the responses to wound injury and repair. There is a balance between inflammation and angiogenesis directed by T lymphocytes that may be a part of the mechanisms that account for tissue repair and scar formation.

[0020] In a study using diabetic and wild type mice, lack of lymphocytes compromises wound healing independent of diabetes. The lack of these cells, even in non-diabetic mice, mimics the phenotype observed in wounds under diabetic conditions. Moreover, the combination of diabetes and the lack of lymphocytes further impairs the wound healing conditions, indicating that when the innate regulatory function is lost in these mice, excessive Ml macrophage polarization, poor angiogenesis, and impaired wound healing are worsened. Application of mature B lymphocytes as found in PBMCs greatly accelerated the healing of acute and chronic wounds in both diabetic and nondiabetic mice. The same healing effect was produced when B cells from older obese diabetic mice were applied to acute wounds in similarly aged, obese diabetic mice. Both groups of animals were equivalent to 55-to 60-year- old morbidly obese patients with uncontrolled diabetes, the most difficult population to treat. The sort of mature B cells used in this study have a limited life span, and once applied on a wound, they remained active at the site for up to 14 days. This makes these cells easier to control than other types of cells used in therapies and makes side effects unlikely. Overall, the presence of B lymphocytes was associated with increased tissue proliferation, reduced cell death, and a more supportive environment for wound healing.

[0021] Recent advances in single-cell transcriptomics have allowed the characterization of gene expression in specific cell types, in environments such as wound healing, which require well-coordinated actions by numerous cell types, including monocyte / macrophages, fibroblasts, endothelial cells, and keratinocytes. Over the last 25 years, innovative therapies have been proposed with the aim of fostering the regenerative potential of multiple immune cell types. This aim can be achieved by promoting cell mobilization into the circulation, their recruitment to the wound site, modulation of their local activity, or their direct injection into the wound. Studies using autologous peripheral blood mononuclear cells (PBMCs) or their proteinderivatives in wound healing have been encouraging. An earlier study showed that topical application of activated PBMCs effectively initiated epithelialization of ulcerated, dermal wounds and that wound closure was present in 92% of patients after 60 days, as compared to standard therapy. In particular, paracrine factors are being considered as a promising option because they provide pro-angiogenic and anti-apoptotic mediators for cell proliferation and migration. To advance Holzinger's "activated PBMC-based therapy," the cell-free secretome of apoptotic PBMCs was studied.

[0022] In a Phase 1 study, topically administered autologous secretome (APOSEC: the derived proteins from the cells) of apoptotic (irradiated) PBMCs in healthy male volunteers with artificial dermal wounds were treated. The PBMC secretome is a mixture of paracrine factors containing multiple pro-angiogenic proteins, lipids, and exosomes. This allogeneic treatment was found to be safe and well tolerated in human intact skin, as well as on the open wound area. This study concentrated on the biological effects of paracrine factors derived from stressed white blood cells. The supernatant provides a potent cell-free alternative, displaying a possible diminished immunogenicity as compared to cell-based therapy. APOSEC stimulates migration of fibroblasts, keratinocytes, and endothelial cells in vitro, which are crucial elements in the physiology of wound healing. Moreover, APOSEC contains significant amounts of antimicrobial peptides that possess antimicrobial activity against opportunistic skin pathogens, especially Escherichia coli and Pseudomonas aeruginosa . With regard to the cataclysmic consequences of bacterial infection for wound regeneration and healing, in severe cases involving non-remediable tissue impairment necessitating amputation, this particular attribute emphasizes the clinical potential of APOSEC. Another approach has been the use of allogeneic gamma-irradiated cord blood mononuclear cells in a patient with critical limb ischemia, which led to improved wound closure and vascularity .

[0023] Addition of mononuclear cells (lymphocytes and monocytes) to platelets can enhance wound healing. The immune system plays an integral role in successful wound healing. In addition to contributing to host defenses, immune cells are critical regulators of wound healing through the secretion of cytokines, lymphokines, and growth factors. Various studies ofPRP technologies reveal that the wide variation of blood components, including platelets, redblood cells, leukocytes, pH, and glucose in PRP extractions play an important role in successful wound healing. The high concentrations of cells are important, as are the white blood cell count in PRP samples. The non-standardized method of recovering a PRP fraction has frequently been ignored by investigators, which is considered insignificant. The lack of standardization of PRP preparations for clinical use has contributed at least in part to the varying clinical efficacy in PRP use.BRIEF SUMMARY

[0024] The current invention enables the separation of mononuclear cells (monocytes and lymphocytes) and platelets from whole blood by centrifugation. According to at least one embodiment of the invention, there is provided a blood collection tube that utilizes a porous foam separator. The foam separator is positioned within the blood collection tube forming a barrier between a liquid density medium, such as Ficoll®Paque , placed below the foam barrier, and a liquid anticoagulant, preferably sodium citrate, which is placed above the foam barrier. The evacuated blood collection tube allows the collection of a blood sample using standard venipuncture prior to centrifugation. The collected blood mixes with the anticoagulant upon blood draw to prevent coagulation of the blood. Upon centrifugation, the blood components are separated by their cell density, allowing the denser red blood cells and granulocyte populations to migrate below the foam barrier, while the less dense mononuclear cells and platelets remain above the barrier. Effective separation and isolation of these cells is often critical to various clinical assays, as well as to research laboratory protocols. Therapeutic application of the isolated cell fractions is also of importance, such as the use of platelet rich plasma in wound care.

[0025] Once the separation has occurred, one exemplary application involves the direct injection of the mononuclear-platelet plasma suspension into the injured site. The upper fraction of plasma, mononuclear cells, and platelets (the mononuclear-platelet rich plasma or M-PRP) is aseptically transferred to an injection device, such as a needle and syringe. The platelets collected in PRP are activated by the addition of calcium gluconate, as an example, which induces the release of factors from alpha granules. The process increases theconcentration of mononuclear cells and platelets and the concentrated M-PRP is then injected into and around the affected area, jump-starting and significantly strengthening the body's natural healing signals.

[0026] Another similar application is the use of the M-PRP suspension as a "glue" for split thickness skin grafts. This application helps fasten the graft to the substrate and the cells contained within the M-PRP speed up the "take" of the graft. This use also eliminates the need for stitches or staples in order to fasten the graft to the substrate.

[0027] According to at least one other application, and once the separation has occurred, the upper fraction of mononuclear cells, platelets, and plasma is aseptically transferred to a second evacuated tube containing calcium chloride. Upon contact, the calcium ions overcome the anticoagulant effect of the citrate and cause the plasma fibrinogen to activate to fibrin, causing the cell suspension to be trapped in a fibrin clot. Behind this clot formation, there is the intrinsic coagulation pathway, which is activated at the level of factor XII by the tube glass surface and proceeds in the presence of calcium to convert prothrombin to thrombin, subsequently fibrinogen to fibrin, and consequently facilitates fibrin polymerization and cross-linking.

[0028] The mechanism of Ca2+-induced clot formation can include a fibrin mesh deposited on platelet aggregates in a white thrombus. Platelet aggregates function like nuclei of clot formation and are located mainly near the center or in a deep region of a clot, and this clot may be classified essentially as a white thrombus. In this case, growth factors stored in platelet a granules can be assumed to be retained for a relatively long time. This type of clot functions as a long-lasting carrier with a superior regenerative potential.

[0029] As clot formation proceeds, the second evacuated tube is centrifuged to produce a solid mononuclear-platelet matrix (MPM), which can be placed directly on a wound following centrifugation. This fibrin matrix allows easier handling of the MPM and can be sutured into place if required, as in certain orthopedic applications.

[0030] According to at least one application, once the separation has occurred, the upper fraction of mononuclear cells, platelets, and plasma is aseptically transferred to a second evacuated vial containing calcium chloride. This vial can have a flat bottom, which upon centrifugation forms the MPM into a flat membrane. This MPM membrane has a greater surface area and thus there is greater exposure of the wound bed to the MPM.

[0031] A "ready to use" kit according to at least one embodiment of the invention comprises a sealed container containing calcium chloride as coagulation activator. Calcium chloride activates the fibrinogen present in patient's plasma when this is introduced into the sealed container. The kit, according to the invention, has the great advantage of allowing the preparation of autologous fibrin glue which may be used with no risk of viral infections or rejection cases. Another advantage of the kit according to the present invention is that it allows the preparation of autologous fibrin glue from patient's plasma in a very short time and in the desired form of clots or membrane or spray. Still another advantage of the ready to use kit, according to the present invention, is to allow the autologous fibrin glue to be obtained at costs proportionally lower with respect to the known systems.

[0032] These and other technical features and advantages will be readily apparent from the following Detailed Description, which should be read in conjunction with the accompanying drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 Illustrates a sodium citrate-based anticoagulant deployed within an evacuated blood collection tube and separation assembly; and

[0034] FIG. 2 is an exploded assembly view of a transfer device made in accordance with aspects of the present invention.DETAILED DESCRIPTION

[0035] The following describes one or more exemplary embodiments of a method and related apparatus in accordance with aspects of the present invention. In accordance with oneexemplary embodiment , a sodium citrate-based anticoagulant solution is prepared in the following manner. First, trisodium citrate-2H2O and citric acid- H2O are dissolved in water in amounts sufficient to yield a sodium citrate solution having a desired concentration and pH which fall within the ranges set forth below. For example, a 0.1M sodium citrate solution having a pH of 7.0 (+0.15) can be prepared by dissolving 29.4 grams of Na citrate-2H2O and 0.27 grams of citric acid-H2O in a liter of H2O. The concentration of the sodium citrate-based solution should be sufficient for preventing coagulation of a blood sample either added to a blood separation / collection device or involved in some other laboratory / clinical technique. In particular, the concentration of sodium citrate should range from about 0.05M to about 0.2M, and preferably from about 0.08M to about 0.13M. A most preferred range is from about 0.09M to about 0.11M. Additionally the pH of the final sodium citrate-based solution ranges from above pH6.0 to about pH8.5, and preferably from about pH 6.5 to about pH7.5. In a most preferred embodiment, the pH ranges from about pH6.85 to about pH7.15. Once the solution of the present invention has been prepared according to the aforementioned steps, the solution may be either employed in some laboratory technique or added to any of several blood separation and / or collection tubes available in the art for separating lymphocytes, monocytes, and platelets from heavier phases of whole blood or a pre-treated cell fraction thereof. While the sodium citrate-based anticoagulant solution of the present invention can be used for any blood separation device, the herein described solution potentially affords the greatest advantages when used with those devices utilizing a foam separation layer employed as a barrier for isolating various components of the device prior to centrifugation. In particular, the solution of the present invention can be employed in the construction of an improved blood separation assembly. A preferred embodiment of the blood separation assembly includes a container having a closed end and an open end. The container is preferably of the type known in the art that is capable of collecting a blood sample, which is typically tubular in shape, and further being capable of undergoing subsequent centrifugation for separation of the contained sample components.

[0036] Referring to FIG. 1, an exemplary blood collection and separation assembly 100 is shown. The blood collection and separation assembly 100 includes a container or tube 112 and a foam barrier layer 114, the latter being positioned within the tube 112 at a first position.

[0037] A variety of specific foam compositions known in the art may be used in the tube 112, depending upon the desired operation to be performed. For example, if the foam barrier layer 114 is employed as a separation medium as well as a barrier, the foam barrier layer 114 should have a porosity and hydrophobicity that prevents the premature mixing of the liquid anticoagulant and the liquid density medium, but allows the movement of blood cells through the foam barrier layer 114. In such an assembly, the foam barrier layer 114 maintains isolation of a blood sample delivered to the tube 112 from the liquid density gradient material residing in the tube 112 until analysis can be performed later. In such a situation, the porosity of the foam barrier layer 114 should be within a sufficient range for allowing adequate separation of the mononuclear and platelet cell layers from the other components of the blood sample.Characteristic foams are well-known in the art and are typically water insoluble and chemically inert to blood. A preferred embodiment of the improved blood separation assembly of the present invention also includes a suitable liquid density separation medium 116, the latter being employed within the container or tube 112 at a second position, which is further away from the open end of the container 112 than the foam barrier layer 114.

[0038] Still referring to Fig. 1, an exemplary liquid density separation medium 116 is shown positioned immediately below the foam barrier 114 in accordance with this embodiment. Typically, the liquid density separation medium 116 will be of a suitable type known in the art for separating mononuclear cells and platelets from whole blood, a nonlimiting example of such being Ficoll-Paque™.

[0039] Upon the subsequent centrifugation (not shown) of the herein described separation assembly 110, the foam barrier layer 114 maintains its original position, but as centrifugation proceeds, the red blood cells and granulocytes gradually separate from the mononuclear cell and platelet fraction, and which becomes concentrated in a layer immediately above the foam barrier layer 114. The red blood cells and granulocytes migrate through thefoam barrier layer 114 to displace the liquid density separation medium 116 below. As the liquid density separation medium 116 is displaced, the medium 116 moves upward through the foam barrier layer 114 to mix with the mononuclear cell and platelet fraction / anticoagulant solution. Red blood cells and granulocytes are pelleted toward the bottom of the tube 112, while the lymphocytes, monocytes, and platelets form a highly purified mononuclear-platelet cell layer immediately above the foam barrier layer 114, thereby facilitating isolation and subsequent removal of the mononuclear and platelets cells.

[0040] Finally, an anticoagulant sodium citrate-based anticoagulant solution 118 according to this exemplary embodiment is positioned above the foam barrier layer 114 so that the anticoagulant solution 118 may adequately contact a whole blood sample (or pre-treated cell fraction thereof) introduced into the tube 112 for centrifugation and subsequent isolation of the mononuclear and platelet cell layer. Referring again to FIG. 1, the anticoagulant solution 118 is shown positioned above the foam barrier layer 114 in closer proximity to the open end of the separation tube 100 than the foam barrier layer 114. As previously mentioned, the anticoagulant solution 118 should have an effective concentration of sodium citrate sufficient for preventing coagulation of a sample of blood when such sample is later added to the tube 112 for centrifugation and subsequent analysis. The anticoagulant solution 118 should have a pH ranging from above pH6.0 to about pH8.5, and preferably from about pH6.5 to about pH7.5. Most preferably, the pH of the anticoagulant solution 118 should range from about pH6.85 to about pH7.15. Optimally, the pH should be 7.0. In this exemplary embodiment of the present invention, the concentration of sodium citrate in the anticoagulant solution 118 should range from about 0.05M to about 0.2M, and preferably from about 0.08M to about 0.13M. Most preferably, the concentration of sodium citrate should be from about 0.09M to about 0.11M. Optimally, the concentration should be 0.1M.

[0041] While the anticoagulant solution 118 of this specific embodiment is primarily composed of sodium citrate, additional reagents may be added, such as cell-sustaining solutions or other reagents, to provide additional properties to the anticoagulant solution. This embodiment also includes a free space 200 adjacent to the open end of the container or tube 112 which is of a sufficient volume to receive a sample of whole blood or a fraction thereof,either alone or in conjunction with an added reagent. In particular, FIG. 1 shows the free space200 positioned above anticoagulant solution 116 in order to provide suitable space for accommodation of a blood sample to be separated.

[0042] Additionally, the assembly of the present invention may optionally include a closure means for sealing the open end of the container or tube 112. Typically, the closure means will be suitable for providing a vacuum seal of the open end of the container or tube 112, as well as being pierceable such as a septum, by a needle in order to adapt the container 112 for drawing a sample of blood from a test subject. Again, referring to Fig. 1, a pierceable closure 122 is provided in the open end of the container or tube 112 for creating a vacuum sealing of the container as mentioned above. Upon the addition of a blood sample to the assembly 100, mixing of the sample with the anticoagulant solution 118 occurs, typically by manual inversion of the container 112. The foam barrier layer 114 remains in a fixed, first position in the tube 112 to serve as a barrier isolating the blood sample / anticoagulant solution suspension from any contact with the other components of the assembly, such as the liquid density separation medium 116.

[0043] The present invention further includes a method for separating lymphocytes, monocytes, and platelets from more dense phases of a sample of whole blood or a pretreated cell fraction thereof. The method may, according to one or more embodiments include the steps of providing a container having an open and a closed end. Preferably, the container is a blood collection / separation tube of the type mentioned above. The method includes introducing a first layer of a density gradient liquid, a foam barrier in the container or tube at a second position. The method further includes introducing an anticoagulant solution into the container at a third position in closer proximity to the open end of the container than the second foam barrier position. The method also includes the steps of limiting the pH of the solution to any of the preferred ranges previously mentioned as well as the step of introducing the anticoagulant solution of the present invention into the container with an effective concentration of sodium citrate sufficient for preventing coagulation of a blood sample.Additionally, the method includes the step of limiting the concentration of sodium citrate in thesolution to any of the pH ranges previously mentioned in the description of the anticoagulant solution.

[0044] The method of the present invention also includes the step of introducing a sample of whole blood, or a pretreated cell fraction of blood, or a bone marrow aspirate into the container and the subsequent step of centrifuging the container to induce separation of lymphocytes, monocytes, and platelets from denser phases of the sample.

[0045] Many methods and systems require the transfer of a fluid from one container to another. A common practice is to remove closures on the two containers and to pipette liquid in one container to the other. This practice, however, exposes a contained sample to environmental contaminants. For example, this latter technique is used to transfer plasma that has been separated from red blood cells in a blood sample. A special technique is required, however, to remove the plasma at the interface meniscus. Frequently the high-density, undesirable, lower-fraction red blood cells contaminate the aspirated sample. To avoid this problem, the pipette is frequently maintained a safe distance from the meniscus (i.e., the interface between the plasma and red blood cells), thereby resulting in an incomplete transfer of the sample. The incomplete transfer of the desirable fraction results in lower than optimum volume yield and non-stoichiometric ratios of the sample reagents and those in the second container. This second condition can be a serious source of performance variation of the product. This is the case in many enzyme reactions in which reaction rates are a maximum at certain stoichiometric ratios and rapidly diminish at higher or lower ratios.

[0046] Wound care is one of the most important issues in medicine, especially with respect to chronic ulcers. This issue is important not only because of the high cost of management, but also because of the variable success rate. Other problems associated with wound care include loss of liquids and the possibility of infections occurring. Synthetic or animal-origin membranes have been used in wound care as a dressing or to separate bone cavities from soft tissues in the process of re-ossification.

[0047] One treatment for wound care may include applying biological tissues or sponges (generally protein based) of animal origin, e.g., collagen, fibrin, albumin to a wound site. However, allergic and immunological responses are common with these applications. Fiftypercent of these cases are not resolved with a single application. More than twenty percent may not be resolved even after two applications.

[0048] Another treatment includes skin transplantation, which is performed for the most difficult cases. Skin transplantation is expensive, however, and significantly increases overall treatment costs. A mesh of modified animal collagen may be used to support the new autologous tissue. The application is a difficult process that may take up to 20 days for cultivation of dermal tissue, with the possibility of contamination of the device.

[0049] Overall, methods and systems for preparing autologous PRP, or M-PRP, or a solid fibrin matrix which is capable of regenerating tissue in a living organism are desired.According to various aspects, the present invention also provides systems and methods for forming a solid-fibrin matrix or autologous fibrin membrane capable of regenerating tissue in a living organism. In these methods and systems, anticoagulated plasma containing mononuclear cells and platelets is obtained by centrifugation of a blood sample. The transfer device described herein enables the cell-plasma suspension to be transferred to a second container containing calcium-clotting agents and then immediately centrifuged to obtain a stable, dense, autologous mononuclear-platelet fibrin matrix. The transfer devices described herein may also be used to transfer other liquids in other applications. In other words, the methods, transfer devices, and systems described herein enable concurrent centrifugation and coagulation. By using these systems and methods, at least one of the following may be achieved: 1) the sample is manipulated in a manner by which sterility is maintained; 2) the total volume of plasma is transferred to maximize a full yield of a clot; 3) the stoichiometric ratio of anticoagulant and calcium clotting agent is maintained in a narrow range to minimize clotting time; 4) the transfer is completed quickly and can be performed inter-operatively within the half-life of the platelet- derived growth factors; 5) health care providers not normally performing these operations (e.g., Nurse Practitioner) can easily perform these methods and operate the systems; and 6) the devices are single use in order to prevent re-use and possible contamination by blood-borne pathogens.

[0050] Generally speaking, the invention provides integrated systems and methods for preparing a solid-fibrin matrix or autologous fibrin membrane that can be used to regeneratetissue in a living organism. In one exemplary embodiment (shown in FIG. 1), the system comprises a blood collection tube 112 with a pierceable stopper 122 which encloses an evacuated space 120 allowing the collection of a blood specimen. The blood specimen upon entry into space 120 mixes with an anticoagulant solution 118 which inhibits clotting of the blood specimen. Upon centrifugation of the blood collection tube 112, the anticoagulated blood specimen is separated according to specific densities of each component. The denser components move through a porous foam barrier layer 114 and dilute and displace the liquid density medium 116 upward through the foam barrier layer 114. The slightly diluted liquid density medium 116 then contacts the less dense blood components and keeps them above the foam barrier layer 114. The mononuclear-platelet rich plasma (M-PRP) can then be recovered.

[0051] In another embodiment (shown in FIG. 2), the system comprises a primary container 10, a secondary container 14 and a transfer device 18. Preferably, the primary and secondary containers 10, 14 are tubes, or a flat bottom vial 38, and more particularly, test tubes or vials, although any container that is capable of holding a fluid or liquid and being centrifuged is suitable for use in accordance with the present invention. Preferably, the containers 10, 14, and 38 are made from a durable lightweight materials, such as, but not limited to glass or plastics.

[0052] The primary container 10 should be capable of drawing blood therein using standard venipuncture techniques. Preferably the primary container 10 is sealed using the stopper 22 while the blood is being drawn to prevent contamination, although the container 10 may be sealed shortly thereafter. A variety of stoppers 22 can be used to seal the primary container 10, e.g., a rubber stopper, cap, foam, elastomer, or other composite. The stopper 22 should be capable of being pierced or punctured, and therefore rubber or silicone are preferred materials from which the seal is fabricated, although any material that provides a seal and is capable of being pierced such as, using a needle, can be used. The primary container 10 may contain an anticoagulant solution 25. The anticoagulant in the solution 25 preferably comprises a calcium-binding agent. More particularly, the anticoagulant may comprise sodium citrate, ethylenelendiaminetetraacetic acid disodium salt, ethylenelendiaminetetraacetic acid dipotassium salt, or ethylenelendiaminetetraacetic acid tripotassium and combinations thereof.Preferably, the primary container 10 contains a sodium citrate solution, preferably at pH 7.0. The anticoagulant solution 25 tends to slightly dilute the blood collected in the primary container 10 to place the blood in condition for centrifugation. In addition, the primary container 10 includes a density-gradient separation medium 26, air 27 as well as a porous foam barrier layer 28.

[0053] The separation medium 26 may separate red blood cells from a mononuclearplatelet-rich plasma during centrifugation of a blood sample. In one example, the separation medium 26 may be found in the bottom of the primary container 10. Although any densitygradient separation medium 26 capable of separating liquids having different densities during centrifugation is suitable for use with the invention, preferably the phase separation device 28 is a foam barrier, and more preferably, a porous foam barrier layer. This foam barrier layer is coupled with a density gradient fluid to effect separation.

[0054] The transfer device 18 according to this exemplary embodiment may comprise two (2) pieces, as shown (FIG. 2). More specifically, the transfer device 18 comprises a cannula having a first end 42 having a first opening and a second end 50 having a second opening. The first and second ends 42 and 50 of the cannula are sharp or pointed (or may also have a bevel ground on them) to be able to puncture or penetrate the stoppers 22 and 24 of the primary and secondary containers 10, 14 or 38. The cannula is recessed and coaxially mounted within a Luer-Lok access device 58 to prevent accidental finger stick during manipulation of the primary and secondary containers. The access device 58 according to this specific embodiment has two cylindrical, opposed guides 62 and 64, which are centrally and axially oriented with the cannula. Guides 62 and 64 serve to guide the primary and secondary containers 10, 14 or 38 onto the first and second ends 42 and 50 to act as a transfer device 18.

[0055] The first and second ends 42 and 50 of the cannula may be encompassed or covered by safety valves, sheaths or elastomeric sleeves 68 and 72, which form a hermetic seal. The foregoing components 68 and 72 also cover the first and second openings 46 and 54. When the first and second ends 42 and 50 puncture the safety valves, sheaths or elastomeric sleeves 68 and 72, these components 68 and 72 retract accordingly. The first and second ends 42 and 50 extend far enough to fully puncture the stoppers 22 and 24, but not extend much furtherinto the containers 10, 14 or 38. This allows maximum volume transfer of the inverted primary container's 10 liquid volume to the secondary container 14 or 38. The safety valves, sheaths, or elastomeric sleeves 68 and 72 prevent the flow of gas or liquid when not punctured. Suitable materials for the foregoing components 68 and 72 include, but are not limited to, rubber varieties and thermoplastic elastomers.

[0056] Subsequent to centrifugation of the blood collection tube 10, the sealed primary holder 10 is inverted before the transfer device 18 is used to puncture the seal of the stopper 22. In other words, the primary container 10 is inverted such that the sealed opening is in the lowest vertical position. Inverting the primary container 10 changes the order in which the layers are arranged. Above the seal of the stopper 22 are the following layers in sequence from bottom to top: the mononuclear-platelet-rich plasma, the foam barrier layer 14, 114 (see FIG. 1), the residual gas, the separation medium and the red blood cells.

[0057] Next, the secondary container 14 or 38 is placed in a vertical position with its sealed opening 24 in the topmost position as shown in FIG. 2. This configuration positions the secondary container 14 or 38 for the transfer of the primary holder's contents therein. The transfer device's guide 64 is then placed over and guides the secondary container 14 or 38 therein, while the inverted primary container 10 is then placed into the other guide 62 (or vice versa). In other words, either the first or second end 42 or 50 of the cannula can be used to puncture either seal of stoppers 22 or 24. Because the transfer device 18 is symmetrical on either end, the user is provided a degree of foolproof operation. The user then forces the containers together to puncture the seals of both stoppers 22 and 24 with each respective cannula end 42 and 50. The two valve sleeves, sheaths or elastomeric sleeves 68 and 72 covering the first and second ends 42 and 50 further enhance the foolproof operation. First, if the first end 42 punctures the primary seal of the stopper 22 (again, either end can be used to puncture either seal), the unpunctured sleeve 72 covering the remaining end 50 will contain the fluid, thereby preventing the fluid from spilling. On the other hand, if the second end 50 punctures the other seal 24 (and the sleeve 72 accordingly) first, the vacuum is maintained by the sleeve 68 covering the first end 42.

[0058] Once the ends 42 and 50 puncture both sleeves 68 and 72 and seals of the stoppers 22 and 24 as shown, the desired fluid is transferred from the primary container 10 to the secondary container 14, or 38 if it is used, by a pressure differential. In other words, because the pressure in the secondary container 14 or 38 has been evacuated, the contents (more particularly, the mononuclear-platelet rich plasma) of the primary container 10 flow into the secondary container 14, or into 38 if the latter device is used. The pressure in the primary container 10, originally at atmospheric, decreases as the liquid level diminishes and the gas volume expands. At no point, however, is the pressure equal to zero. Because the secondary container 14 or 38 is fully evacuated to a pressure equal to or slightly greater than zero, the pressure therein does not increase as the tube is filled, since there is little or no gas to compress. Accordingly, the apparatus 18 may be used to transfer a wide variety of liquids and solutions from one tube to another and should not be construed to be limited only to the transfer of blood.

[0059] Because of the sequential arrangement of the layers in the primary container 10, the mononuclear-platelet rich plasma is easily transferred. In addition, because the primary container 10 is also preset to an evacuation level, the container only partially fills after blood collection. This allows the gas in the "head space" to remain significantly above zero during transfer when its volume is expanded, thereby allowing fast and complete transfer to the secondary container 14 or 38, if used. This is dictated by the ideal gas law and the Hagen- Poiseuille equation.

[0060] When the transfer of the mononuclear-platelet plasma fraction to the secondary container 14 or 38 is completed, thereby allowing maximum yield and maintenance of the appropriate stoichiometric ratio of reagents. The mononuclear-platelet plasma then contacts the coagulation activator 36 in the second container 14 or in 38, thereby creating a mixture which can be immediately centrifuged to form a solid fibrin matrix. The pressure differential between primary and secondary containers 10 and 14 or 38 is substantially maintained throughout transfer, allowing rapid transfer. The transfer device 18 is unaffected by order of tube engagement, rendering the system virtually foolproof. Finally, the transfer occurs without venting, maintaining sterility and non-contamination of the sample.

[0061] Overall, the transfer device 18 provides a quick and efficient way of contacting the mononuclear-platelet plasma suspension with the calcium coagulation activator 36, immediately after which concurrent coagulation and centrifugation of the plasma can take place in order to form the solid fibrin matrix. The solid fibrin web is suitable for regenerating body tissue in a living organism. Such a method alleviates the need to first pre-concentrate the plasma by removing water therefrom before the plasma is contacted with the calcium coagulation activator 36. In addition, the transfer device 18 can be used to transfer blood or other fluids in a wide variety of application.

[0062] The use of the secondary tube 14 allows the resulting mononuclear-platelet matrix formed following centrifugation to form in the shape of the secondary tube's 14 bottom, thus forming a mononuclear-platelet matrix (MPM). Alternatively, using the secondary vial 38, following centrifugation, the resulting mononuclear-platelet matrix forms a MPM membrane with the same diameter as the secondary vial 38.

[0063] In one embodiment, the invention also provides a ready-to-use kit which comprises the primary container(s) 10, the secondary container 14 or 38, the transfer device 18, an alcohol swab to cleanse the venipuncture site, an elastic tourniquet, a multiple sample blood collection needle set (21 gauge x 1"), a safety holder, an elastic bandage, and a sterile culture dish to receive the MPM matrix or membrane. The components can be arranged in a wide variety of manners within the kit.

Claims

WHAT IS CLAIMED:

1. A method for separating plasma, lymphocytes, monocytes, and platelets from the denser phases of a sample of whole blood or a pretreated cell fraction thereof, the method comprising the steps of:• providing a container having an open end a closed end, the container further having a first layer of a density gradient fluid therein at a first position, a foam barrier contained therein at a second position, and an anticoagulant solution for preventing coagulation of the sample or pretreated cell fraction when the sample or pretreated cell fraction is introduced into the container, the anticoagulant solution being located at a third position within the container in closer proximity to the open end of the container than the second layer, the anticoagulant solution having an effective concentration of sodium citrate with a pH adjusted to modify the pH of the anticoagulant solution, sufficient for preventing coagulation of the sample or pretreated cell fraction when the sample or pretreated cell fraction is added to the container, the anticoagulant solution having an adjusted pH ranging from pH6.5 to pH8.5;• introducing the sample or pretreated cell fraction into the container; and• centrifuging the container to induce separation of lymphocytes, monocytes, and platelets from denser phases of the sample or pretreated cell fraction, wherein the anticoagulant solution affords more efficient cell separation having reduced red blood cell contamination and granulocyte contamination and an enhanced recovery and viability of the separated lymphocytes, monocytes, and platelets.

2. The method according to claim 1, wherein the step of providing the container includes the additional step of introducing the first layer of the density gradient liquid into the container at the first position.

3. The method according to claim 1, wherein the step of providing the container includes the step of introducing the foam barrier into the container at the second position within the container in closer proximity to the open end of the container than the first layer.

4. The method according to claim 1, wherein the step of providing the container includes the step of introducing the anticoagulant solution into the container at the third position within the container in closer proximity to the open end of the container than the second layer, the anticoagulant solution having an effective concentration of sodium citrate sufficient for preventing coagulation of the sample.

5. The method according to claim 4, wherein the step of providing the container includes the step of limiting the pH of the anticoagulant solution to a range from about pH6.5 to about pH7.5.

6. The method according to claim 1, wherein said step of providing the container includes the step of limiting the concentration of the anticoagulant solution to a range from about 0.05M to about 0.20M.

7. The method according to claim 6, wherein the step of providing the container includes the step of limiting the concentration of sodium citrate in the anticoagulant solution to a range from about 0.08M to about 0.13M, preferably an anticoagulant solution to a range of about 0.09 to about 0.11M8. The method according to claim 7, wherein the step of providing the container includes the step of limiting the anticoagulant solution pH to pH7.0 and the step of limiting the concentration of sodium citrate in the anticoagulant solution to 0.1M.

9. The method according to claim 1, further comprising the step of transferring the separated lymphocytes, monocytes, and platelets from the container to a second containerusing a transfer device, thereby allowing the mononuclear-platelet plasma to contact a calcium chloride coagulation activator in the second container, thereby creating a mixture which can be immediately centrifuged to form a solid fibrin matrix.10 . An assembly for separating lymphocytes, monocytes, and platelets from denser phases of a sample of whole blood, or a pre-treated cell fraction thereof, or a bone marrow aspirate, the method comprising:• providing a container having an open end, the container being suitable for centrifugation and having disposed therein a liquid density gradient material, a foam barrier, the sample of whole blood , pre-treated cell fraction, or aspirate and an anticoagulant citrate solution having an adjusted pH ranging from pH6.5 to pH8.5, the anticoagulant solution comprising an effective concentration of sodium citrate and pH adjusted to modify the pH of the anticoagulant solution sufficiently for preventing coagulation of the sample, pre-treated cell fraction, or aspirate; and,• centrifuging the container to induce separation of lymphocytes, monocytes, and platelets from denser phases of the sample, pre-treated cell fraction, or aspirate, wherein the anticoagulant solution affords more efficient cell separation by reducing red blood cell contamination and reducing granulocyte contamination with enhanced recovery and viability of the separated lymphocytes, monocytes, and platelets.

11. The assembly according to claim 10, wherein the density gradient fluid contains a low molecular weight ionic substance and is an aqueous solution with a molecular weight of less than about 1500Da.

12. The assembly according to claim 11, wherein the low molecular weight ionic organic substance is selected from at least one of the group consisting of sodium diatrizoate, derivatives thereof and combinations thereof.

13. The assembly according to claim 11, wherein the density gradient fluid is a high molecular weight organic substance which is a synthetic high molecular weight (MW ~400,000 Da) polymer of sucrose and epichlorohydrin, which is readily soluble in water and derivatives and combinations thereof.

14. The assembly of claim 10, which further comprises a closure for sealing the open end of the container and is suitable for vacuum sealing .

15. The assembly of claim 14, wherein said closure is pierceable by a needle for supplying a blood sample to said container which is adapted to draw said sample.

16. The assembly of claim 10, wherein the sample of whole blood initially in the container is anticoagulated by the liquid citrate anticoagulant contained within the container.

17. The assembly of claim 10 wherein the low molecular weight ionic organic substance is selected from the group consisting of sodium diatrizoate and combinations thereof.

18. The assembly of claim 10, wherein the foam barrier has a porosity to allow red blood cells and white blood cells to pass through under centrifugal force but has sufficient hydrophobicity to prevent aqueous liquids to pass through without centrifugation.

19. The assembly of claim 10, further comprising a transfer device enabling the transfer of said separated lymphocytes, monocytes, and platelets to a second container allowing the mononuclear-platelet plasma to contact a calcium chloride coagulation activator in the secondcontainer, thereby creating a mixture which can be immediately centrifuged to form a solid fibrin matrix20. The assembly of claim 19, wherein the solution of calcium chloride is sufficient to overcome the anticoagulant effect of the sodium citrate.

21. The assembly of claim 19, in which tryptophan or its metabolite (kynurenine) is added to the calcium chloride solution in concentrations sufficient to affect the metabolism of mononuclear cells, (generally in the 50-100 pM range).

22. The assembly of claim 19, in which thromboxane A2 is added to the calcium chloride solution in concentrations sufficient to affect the metabolism of the mononuclear cells, (generally in the lOOpM range).

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