Diethylenetriamine pentaacetic acid for improving platelet count and function of blood products and as alternative anticoagulant

Abstract

Citrate has been used as an anticoagulant because of its calcium chelation capability, but it may have unwanted off target metabolic and inflammatory effects; accordingly, alternative anticoagulants may have benefits compared with citrate. Diethylenetriamine pentaacetic acid (DTPA) is an octadentate chelator that is FDA-approved to treat plutonium, americium, and curium exposure by chelation. In this disclosure, it is demonstrated that a composition comprising DTPA, DPDA-1, functions effectively as an anticoagulant for the storage of whole blood, with an increased platelet count and improved viscoelastic properties compared with citrate composition, although DTPA-composition requires half of the amount of calcium to restore clotting function, which suggest DTPA composition's lowering the risk of post transfusion hypocalcemia in comparison to citrate transfusion. Therefore, DTPA-composition is presented here as an anticoagulant to preserve platelets.

Description

[0001] Attorney Docket No: 15969-034PC0

[0002] Diethylenetriamine Pentaacetic Acid For Improving Platelet Count and Function Of Blood Products And As Alternative Anticoagulant

[0003] STATEMENT OF FEDERAL FUNDING

[0004] This invention was made with government support grant number W81XWH-21-P-0014 awarded by the U.S. Department of Defense. The government has certain rights in the invention.

[0005] BACKGROUND

[0006] 1. Field of invention

[0007] The invention disclosed herein relates to an anticoagulant, which can preserve platelet numbers and coagulation functions during storage period in refrigeration or freezing conditions and at room temperature.

[0008] 2. Background

[0009] Hemorrhage is the leading cause of preventable death on the battlefield, and whole blood is the preferred resuscitation fluid for bleeding patients. Citrate is currently the only FDA-approved anticoagulant for the storage of whole blood for transfusion. Citrate functions as an anticoagulant by chelating calcium, thus rendering the calcium-dependent clotting factors inactive. In addition to chelating calcium, citrate is also an important tricarboxylic acid cycle intermediate and metabolic signal: citrate slows the rate of glycolysis by inhibiting phosphofructokinase, one of the major regulatory glycolytic enzymes.1'4Because red blood cells rely on glycolysis for ATP generation, large amounts of exogenous citrate could theoretically inhibit red blood cell energy production, thus shortening their lifespan in storage. In addition to the effects it may have on red blood cells during storage, citrate may also have negative effects on patients into whom it is transfused. Increasing evidence suggests that citrate promotes inflammation by providing inflammatory substrates, thus giving patients an unnecessary inflammatory load.5'7Similarly, massive transfusion of citrate anticoagulated blood is associated with the development of hypocalcemia, which contributes to the “diamond of death” in bleeding patients.8'10Attorney Docket No: 15969-034PC0

[0010] SUMMARY

[0011] Given these limitations of anticoagulation of whole blood for transfusion using citrate, it seems worthwhile to explore the potential of alternative anticoagulants that may have fewer off-target effects.

[0012] Recently, this laboratory investigated pyrophosphate as an alternative to citrate for storage of whole blood.11While pyrophosphate was able to anticoagulate whole blood and appeared easier to recalcify than citrate, evidence of platelet clumping over time was found, which will limit its utility for whole blood storage where preservation of platelets is a priority.

[0013] It was then reasoned that synthetic chelators would be able to overcome the enzyme inhibition and pro-inflammatory issues presented by citrate, and we set out to identify candidate molecules. Because calcium has a coordination number between 6 and 8, it was reasoned that hexadentate and octadentate chelating ligands would be the most effective in chelating calcium.12Accordingly, the search led us to diethylenetriamine pentaacetic acid (DTPA), an FDA-approved chelating molecule used to treat exposure to plutonium, americium, and curium.

[0014] Thus, the objective in this disclosure is to assess DTPA for anticoagulant activity and compare it with the current standard of care, citrate. Here, evidence was provided for the superiority of DTPA as an anticoagulant compared with citrate in the storage of whole blood.

[0015] Briefly, human whole blood samples were drawn into a syringe, anticoagulated with either citrate or DTPA, and then stored in miniature storage bags at 4°C for 7 days. After storage, blood cell counts and morphology were assessed, and viscoelastic testing and flow cytometry were performed. Both citrate and DTPA resulted in sustained anticoagulation, which was reversed by the addition of calcium, though DTPA-anticoagulated blood required half as much calcium as citrate-anticoagulated blood to reach maximum clotting activity. Platelet counts were higher in DTPA samples than citrate samples. Viscoelastic testing showed that samples anticoagulated with DTPA demonstrated a lower R-time (10.90 ± 3.38 minutes vs. 13.33 ± 3.02 minutes; p=0.0029) as well as K-time (3.04 ± 1.04 minutes versus 4.64 ± 1.58 minutes; p=0.0079) while alpha angle was increased (52.2 ± 8.74 degrees vs. 41.55 ± 11.43 degrees; p=0.0173) compared with citrate.

[0016] Overall, the clotting potential is increased after recalcification in DTPA treated blood compared with citrate treated blood since the blood stored with DTPA loses less platelets andAttorney Docket No: 15969-034PC0

[0017] consequently loses less clotting potential over time, and DTPA treated blood formed clots faster and more completely compared with citratc-trcatcd blood.

[0018] These results demonstrate that DTPA functions effectively as an anticoagulant for the storage of whole blood, with an increased platelet count and improved viscoelastic properties compared with citrate. It is concluded that DTPA is a promising candidate anticoagulant to replace citrate for blood storage.

[0019] In summary, it is demonstrated here that DTPA surprisingly improved platelet count and coagulation parameters -the faster R and K times and the higher alpha angle- compared with citrate since nothing about DTPA or any other anticoagulants allow to hypothesize that it would have these effects. Outside of the specific phenomenon of EDTA-induced thrombocytopenia, there are no widely recognized differential effects on platelet count between the different anticoagulants that are currently in use today - e.g., EDTA, heparin, and citrate. These results indicate that anticoagulants are not, in fact, as interchangeable as implied in various prior art.

[0020] In one aspect, a composition for blood product preservation in refrigeration or freezing conditions and at room temperature is provided, which comprises diethylenetriamine pentaacetic acid (DTPA), optionally pentasodium DTPA, as an anticoagulant, pH buffer (e.g., monobasic sodium phosphate monohydrate), sugar-source (e.g., dextrose anhydrous), and adenine to make a composition DPDA-1. The composition is a solution to be 5-fold to 20-fold, optionally about 10-fold diluted in a blood product suspension. For example, the concentration of DTPA is 0.5-2% w / v, optionally about 1% w / v (about 20 mM), to make a final concentration of DTPA of 0.025-0.4% w / v, optionally about 0.1% w / v (2 mM) in a blood product suspension. The composition is optionally packed in a bag or container selected from syringe, tube, vial or ampule for blood product collection.

[0021] The blood product that can be contemplated includes whole blood, red blood cells (RBCs), platelets, or combination thereof, in order to treat a subject in need of whole blood-, RBCs-, and / or platelet transfusion. If need be, RBCs are suspended in a red blood cell preservation solution comprising adenine, dextrose, sorbitol, sodium chloride, and mannitol (e.g., ADSOL®). Platelets may be suspended in plasma, a platelet additive solutions (PAS), which is a buffered salt solution containing ingredients like sodium chloride, citrate, acetate, phosphate, potassium, and magnesium, or other physiological salt solutions such as Frog’s Ringer’s solution, mammalianAttorney Docket No: 15969-034PC0

[0022] Ringer’s solution, Ringer’s lactate solution, Krebs Henseleit salt solution, Tyrode solution, Ringer lockc solution, and De Jalon solution and packed in a bag or a container with a volume capacity of 1-1000 mL. In particular, the blood product is platelets.

[0023] In another aspect, a use method for the composition for preserving platelets is provided, which comprises: a step of immediately (c.g. within 1-120 min) adding and gently mixing the solution comprising an effective amount of the composition of claim 1 to platelet suspension; and a step of refrigerating or freezing the platelet suspension with the composition at a temperature of 4°C or below. In that case, the volume of the solution comprising the composition to be added to the platelet suspension is for 5-fold to 20-fold, optionally 10-fold dilution in the final suspension volume.

[0024] In another aspect, a bag or container for the collection of whole blood, RBC, or platelet comprising the solution of the composition is provided. In the bag, the volume of the solution comprising the composition is less than 20%, optionally less than 10% of the total volume of the bag or container. As an example of such bags, a blood collection double, triple, quadruple or quintuple bag system, as known in the art (e.g. HK Medikal), can be implemented, in which the main bag, RBC, or platelet collection bag comprise a solution comprising the composition.

[0025] In another aspect, a test tube, in which DTPA anticoagulant is coated on the internal surface of the tube is provided.

[0026] BRIEF DESCRIPTION ON THE DRAWINGS

[0027] Figure 1. Chemical structure of DTPA

[0028] Figure 2. Study design. Differences between paired samples were analyzed using a paired t-test Figure 3. White blood cell (WBC), red blood cell (RBC), and platelet (PLT) count comparison in CPDA- and DPDA-1 (mixture of DTPA, phosphate, dextrose and adenine) treated human whole blood after 7 days of storage.

[0029] Figure 4. Viscoelastic testing comparison in CPDA- or DPDA-1 treated human whole blood. Figure 5. Platelet count and R-time comparison in citrate- or DTPA treated human whole blood.Attorney Docket No: 15969-034PC0

[0030] Figure 6. Histological assessment. Human whole blood anticoagulated with CPDA-1 (Panel A) or DPDA-1 (Panel B). Normal platelet count and morphology (red arrows) were observed in both groups. Altered neutrophils (green arrow) and echinocytes (black arrows) were seen consistent with the storage time. Magnification: 40x. Stain: HemaDiff, StatLab, McKinney, TX.

[0031] Figure 7. An example of a blood bag for pretreatment with DTPA

[0032] DETAILED DESCRIPTION

[0033] A. Definitions

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements are subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration and any other measurements, quantities or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0035] As used herein, the term “about” means plus or minus 20 percent of the recited value, so that, for example, “about 0.125” means 0.125 ±0.025, and “about 1.0” means 1.0 ±0.2.

[0036] As used herein, the term “octadentate chelating ligand” refers to 1,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetate (DOTA) and diethylenetriamine pentaacetate (DTPA). In coordination chemistry, denticity refers to the number of donor groups in the ligand molecule that binds to the central metal ion in a coordination complex. If only one atom in the ligand binds to the metal atom / ion, the denticity is one (monodentate or unidentate). If the ligand has more than one ion binding to the metal atom / ion, it is called polydentate or multidentate. For example, a hexadentate ligand in coordination chemistry is a ligand that binds to a central metal atom / ion with six bonds. Examples of hexadentate ligands are N,N,N',N'-tetrakis(2-pyridinylmethyl)- 1 ,2-ethanediamine (TPEN) and ethylene diamine tetra acetic acid (EDTA).Attorney Docket No: 15969-034PC0

[0037] As used herein, the term “platelets” (or thrombocytes) refers to the small fragments (minute discs of 1 to 4 pm in diameter) of megakaryocytes produced in the bone marrow. The normal concentration of platelets in the blood is between 150,000 and 450,000 / pL. It has a half-life of only 8 to 12 days in the blood. The principal function of platelets is to form blood clots. When a vascular wall is damaged, the damaged vascular wall activates platelets, which secrete adenosine diphosphate, thromboxane A2, and platelet-activating factor, among others, and recruit more and more additional platelets, thus forming a platelet clot to plug the blood vessel. Then during the subsequent process of blood coagulation with prothrombin / thrombin, fibrinogen / fibrin monomer / fibrin fibers, which are produced in the liver and found in the plasma, fibrin threads form. These threads tightly enmesh the platelets, thus enhancing the plugs. Thus, a reduction in platelet number can cause systemic bleeding. A platelet count below 100,000 / pL is generally considered thrombocytopenia. There are four main causes for thrombocytopenia: (1) Decreased production of platelets due to depressed bone marrow or megakaryocytes (e.g., aplastic anemia, certain leukemia, disseminated cancer, granulomatous disease, myelodysplastic syndrome, or HIV); (2) Decreased platelet survival due to immunologic destruction (e.g., chronic or acute immune thrombocytopenic purpura, systemic lupus erythematosus, B-cell lymphoid neoplasms, post-transfusion or neonatal alloimmunity, drug or infection associated, or non-immunologic destruction of platelets such as disseminated intravascular coagulation and thrombotic microangiopathies), (3) increased platelet sequestration in the spleen due to spleen enlargement, i.e., from a normal level of platelet sequestration 30-35% to 80-90%, and (4) Dilution due to transfusion since with prolonged blood storage, the number of viable platelets is reduced.

[0038] As used herein, the terms "subject" which is used interchangeably with "patient" refers to any mammals, including humans, which are in need of blood transfusion or blood component transfusion such as red blood cells, white blood cells, platelets, plasma, etc.

[0039] As used herein, the term "administering" and its cognates refers to introducing an agent to a subject, and can be performed using any of the various methods or delivery systems for administering agents or pharmaceutical compositions known to those skilled in the art. Various modes of administration are described in Remington: The Science and Practice of Pharmacy 23rd Edition. Preferably, the administration route according to the embodiments of this disclosure is parenteral route, preferably intravenous injection or infusion route.Attorney Docket No: 15969-034PC0

[0040] As used herein, the term "treatment" or “treat” in the context of medical meaning refers to intervention of disease, disorder, condition or one or more symptoms thereof to obtain a desired physiological and / or clinical effect for purposes such as: inhibiting the disease, disorder, condition, or one or more symptoms thereof; slowing or delaying the progress of the disease, disorder, condition, or one or more symptoms thereof; stabilizing (i.e., not worsening) a state of the disease, disorder, condition, or one or more symptoms thereof; and relieving, palliating, alleviating, or ameliorating the severity of the disease, disorder, condition, or one or more symptoms thereof; or preventing remission, whether partial or total and whether detectable or undetectable. “Treatment,” or “treat” may not necessarily indicate complete eradication or cure of disease, disorder, condition or associated symptoms thereof.

[0041] As used herein, the term “composition” refers to a pharmaceutical composition, meaning a mixture of substances suitable for administering to an individual, which includes one or more pharmaceutically active ingredients.

[0042] As used herein, the term “thromboelastography (TEG)” refers to a test to assess blood coagulation by measuring the viscoelastic properties of whole blood, including information about clot formation time, strength, and stability. TEG examines the dynamic process of clot formation, including initiation, propagation, and fibrinolysis, essentially assessing the overall function of the coagulation cascade and platelet activity within the blood sample. Parameters to be analyzed include: (1) R time (Reaction time), which is time taken for the first detectable clot formation; (2) K time (Kinetic time), which is time taken to reach a certain clot strength; (3) alpha angle representing the speed of clot formation, (4) maximum amplitude (MA) representing the maximum clot strength achieved; and (5) Ly30 (Lysis at 30 minutes), which is percentage decrease in clot strength after 30 minutes, indicating fibrinolysis activity. Clinical applications of TEG includes: (1) monitoring bleeding risk in surgery and assessing the need for blood product transfusion during surgery, (2) managing patients on anticoagulation therapy and evaluating the effect of anticoagulants like heparin, (3) diagnosing bleeding disorders such as deficiencies in clotting factors or platelet dysfunction, and (4) assessing bleeding risk in trauma patients.

[0043] As used herein the term “anticoagulant” refers to an agent or drug that prevents or reduces the formation of blood clots (thrombosis). Anticoagulants work by inhibiting various factorsAttorney Docket No: 15969-034PC0

[0044] involved in the coagulation cascade, the process by which blood forms clots. Anticoagulants are used to prevent thrombosis during or after surgery or other procedures or for the treatment of conditions such as deep vein thrombosis (DVT), pulmonary embolism (PE), stroke or existing blood clots. There are several types of anticoagulants, including indirect thrombin inhibitors (e.g., heparin), warfarin and other coumarin anticoagulants, oral direct factor Xa inhibitors, direct thrombin inhibitors. Also, calcium-chelating agents such as citric acid, oxalic acid, EDTA, EGTA, DTPA, and DCTA (trans- 1,2-diamino cyclohexane-N,N,N',N'-tetraacetic acid) are considered anticoagulants in that they prevent blood from clotting by chelating calcium ions, which are essential for the clotting process.

[0045] B. Overview

[0046] On objective leading to the discovery of embodiments described herein was out to evaluate DTPA, formulated as DPDA-1 (DTPA, phosphate, dextrose and adenine), as an anticoagulant for the storage of whole blood. After 7 days of cold storage, viscoelastic testing confirmed that DPDA-1 functions as an anticoagulant. Furthermore, platelet count was improved in DPDA-1 samples compared with citrate anticoagulated blood while TEG results suggested superior clotting function with less calcium needed for these samples.

[0047] Recently, pyrophosphate was examined as a potential candidate to replace citrate as the anticoagulant to store whole blood.11While those samples remained anticoagulated after a limited period of storage in tubes, platelet clumping was observed. In the current study, DTPA was tested under the more clinically applicable conditions of refrigerated bag storage. In these conditions, DPDA-1 demonstrated sustained anticoagulation while preserving platelet count and function better than CPDA (a cocktail of citrate, phosphate, dextrose and adenine)- 1. It is believed the preservation of platelet count and function is important in whole blood storage, as transfusion of whole blood units is increasingly seen as beneficial in the treatment of severely bleeding patients.14,15

[0048] Viscoelastic testing demonstrated a faster onset of coagulation after recalcification for DPDA-1 samples as indicated by lower R- and K-Times. Additionally, the maximum amplitude, which serves as an indicator for platelet count and function, trended towards a statistically higher clot strength in DPDA-1 samples but did not reach statistical significance (p=0.0523). While thisAttorney Docket No: 15969-034PC0

[0049] study was powered based on preliminary findings for platelet count, it was believed that a higher sample size would have also resulted in a statistically significant difference in clot strength as indicated by the maximum amplitude. These findings indicate that DPD A- 1- anticoagulated whole blood might be able to contribute to adequate coagulation after transfusion to a greater extent than CPDA- 1 treated whole blood.

[0050] Interestingly, viscoelastic testing indicated that clotting in DPDA-1 samples was superior to CPDA-1 samples while only 50% of the calcium dose was needed. This suggests that DPDA-1 -anticoagulated whole blood might lower the risk of post transfusion hypocalcemia in comparison to citrate transfusion. Calcium levels can be significantly decreased with rapidly transfused blood products due to the citrate preservative that is added. Citrate binds to the patient's endogenous calcium when blood products are administered, rendering calcium inactive. As a result, undesirable physiological effects can occur. While citrate is cleared rapidly in healthy humans, its clearance is impaired in critically ill or injured patients.8This increases the risk for hypocalcemia, which contributes to mortality and morbidity in bleeding patients.9 10

[0051] DTPA is already approved by the FDA as an intravenous chelating agent for plutonium, americium, and curium exposure. For healthy adults, a dose of 0.5 - 1 g Ca-DTPA is recommended as an intravenous bolus within the first 24 hours after contamination which can be continued daily until the desired effect is reached.16However, after 24 hours Zn-DTPA instead of Ca-DTPA should be used, as Ca-DTPA chelates trace metals, in particular zinc, more effectively than Zn-DTPA. Depletion of zinc levels by DTPA might lead to altered DNA synthesis, which led to fetal but not maternal toxicity in animal studies.17

[0052] C. Embodiment Examples

[0053] Anticoagulants are drugs that prevent blood clot formation both in vitro and in vivo, and they are commonly added to the blood collected in lab tubes to maintain the fluid state of blood for various hematological testing and clinical chemistry analyses. For blood clotting, calcium is essential for enzyme activities of the coagulation cascade, and its removal by chelators such as EDTA, heparin, or citrate prevents blood clotting in the blood collected.

[0054] DTPA has been used as a chelator to treat exposure to plutonium, americium, and curium in vivo. In this disclosure, a new use of DTPA is suggested, which can be added as an anticoagulantAttorney Docket No: 15969-034PC0

[0055] to collected whole blood or blood products such as RBC and platelets for transfusion and as an agent to preserve platelet numbers and functions. The conventional shelf life of whole blood in a refrigerator is about 35 days, RBCs about 42 days, and platelets about 5 days at room temperature. Disclosed herein is a composition comprising DTPA and use method thereof are provided for improving the shelf-life of collected whole blood and platelets as well as their functions after transfusion.

[0056] In one embodiment, a composition comprising DTPA is provided as an anticoagulant for collected blood product preservation, in particular platelets, in refrigeration or freezing conditions and at room temperature. The solubility of DTPA in water at 20°C is about 5g / L. For better solubility, sodium salt forms such as tetra- or penta- sodium DTPA may be considered. The composition further comprises pH buffer, sugar source, and adenine.

[0057] The composition can be in the form of a solution to be 5-fold to 20-fold, optionally 10-fold diluted in a blood product suspension, but dry powder mixture to be dissolved in a certain volume of liquid (e.g., Ringer’s solution, lactated Ringer’s solution, Krebs-Ringer solution, saline, and phosphate buffer solution, optionally without CaCh in the solution) may also be used.

[0058] In a specific example, the concentration of DTPA in a composition solution before dilution is 0.5-2% w / v, optionally about 1% (about 20 mM), to make a final concentration of DTPA of 0.025-0.4% w / v, optionally about 0.1 % (about 2 mM) in a blood product suspension In a specific example, the pH of the composition solution is empirically determined to be about 5.9, and there are several pH buffers for this pH range, including histidine (pH 5.5-7.4), maleate (pH 5.5-7.2), MES (pH 5.5-6.7), bis-Tris (pH 5.8 -7.2), and phosphate (pH 5.8-8.0). In a certain embodiment, the pH buffer is monobasic sodium phosphate monohydrate of 10-25 mM. However, higher neutral pH range (pH 6.0- pH 7.6) and pH buffers for such pH range may be considered.

[0059] In a certain embodiment, the sugar source is dextrose anhydrous of 150-200 mM. Dextrose (D-glucose) is a fast-acting form of glucose. Maltose, made of two glucose units, can also be considered.

[0060] Adenine is a purine base that can be converted into adenosine triphosphate (ATP), which can be utilized by platelets when they are stored, and thus adenine in the blood product preservation solution helps platelets to maintain their ATP level, delaying a decline in function and improvingAttorney Docket No: 15969-034PC0

[0061] the lifespan of stored platelets. In a certain embodiment, adenine in the DTPA composition solution is 0.5-5 mM.

[0062] In a specific aspect, the osmolarity of the DTPA composition solution is within a range of 270-330 mOsm / L, optionally 280-300 mOsm / L.

[0063] The solution or dry powder mixture may be sterilized before use by irradiation, heating or filtering with 0.2 pm or smaller pore size sterilizing filters.

[0064] In a particular embodiment, the composition comprises DTPA pentasodium salt about 20 mM (about 1% w / v), monobasic sodium phosphate monohydrate about 16 mM, dextrose anhydrous about 170 mM, and adenine about 2 mM to make DPDA-1 composition solution (Table 2), which is an example of the composition. Other higher or less concentrations of the components are within the scope of this invention.

[0065] In a certain embodiment, a method for preserving platelets is provided, which comprises: a step of immediately (e.g. within 1-120 min) adding and gently mixing an effective amount (i.e., an amount that is sufficient to avoid or reduce blood coagulation and / or to preserve platelet count number during blood collection and storage) of DTPA composition solution to platelets collected and suspended in plasma, a platelet additive solutions (PAS), or other physiological salt solutions such as Frog’s Ringer’s solution, mammalian Ringer’s solution, Ringer’s lactate solution, Krebs Henseleit salt solution, Tyrode solution, Ringer locke solution, and De Jalon solution ; and a step of refrigerating or freezing the platelet suspension solution at a temperature of 4°C or below. The volume of DTPA composition solution such as is described above to be added to the platelet suspension is one fourth to one nineteenth of the platelet suspension for 5-fold to 20-fold dilution in the final suspension volume. In a specific embodiment, a ten-fold dilution is provided. Also, such method of use can be applied for the preservation of whole blood or RBCs.

[0066] For better convenience of use, DTPA composition solution or dry powder mixture can be prepacked in a collection bag (Figure 7) or container such as syringe, tube, vial or ampule to be used for whole blood, RBC or platelet collection. The bag or container is made of any suitable materials for blood collection, including but not limited to, polyolefin or polyvinyl chloride (PVC) that is plasticized with triethyl hexyl trimellitate, butyryl-tri-hexyl citrate, or the like. In a collection bag or container, in the case of a DTPA composition solution, the volume of theAttorney Docket No: 15969-034PC0

[0067] prepacked DTPA composition solution is less than 20%, and optionally less than 10% of the total volume capacity of the collection bag or container. The volume of the bag or container can vary, from 100 mL to 1000 mb, depending on the need, but optionally the volume of one blood unit, i.e., about 500-600 mL.

[0068] If the collection bag or container is for whole blood or RBC collection, it may further comprise mannitol (about 5.25 g / L) to maintain osmotic pressure. Mannitol is a cell-impermeant, nonmetabolized, 6-carbon sugar alcohol. When added to the storage solution, it creates a hypertonic environment, drawing water out of the RBCs and preventing them from swelling excessively and hemolyzing (rupturing) during storage, thus preserving their function and viability.

[0069] As an example of a collection bag with prepacked DTPA composition (solution or powder), blood collection double / triple / quadruple / quintuple bag system can be considered, where platelets are separated through centrifugation and filtration process. In such a multi-bag system, the main bag and / or other bags including RBC or platelet collection bag may comprise DTPA composition as an anticoagulant. In addition, an RBC or platelet collection bag in apheresis can contain DTPA composition. Apheresis is a medical technology in which the drawn blood from a person is passed through an apparatus that separates out blood components by centrifugation, such as RBCs (erythrocytapheresis), plasma (plasmapheresis), platelets (plateletpheresis), and leukocytes (leukapheresis), and returns the remainder to the circulation. The component to be removed can be selected by moving the level of the aspiration device.

[0070] Additionally, a DTPA composition may be used in a regional surgery, for example as a regional anticoagulant for medical procedures like Continuous Venovenous Hemodiafltration (CVVHDF), also known as Continuous Renal Replacement Therapy (CRRT).

[0071] Another aspect provided herein is the use of DTPA as an anticoagulant for analytical blood tests, like EDTA, citrate, and heparin, which are currently used today. A solution of DTPA can be spray-dried onto the internal surface of a lab / blood test tube before use or added to the blood sample immediately after putting the sample into the test tube. For example, the anticoagulant property of DTPA can be utilized by coating the internal surface of a test tube with DTPA.

[0072] DTPA has at least a couple of advantages over citrate. First, DTPA has better calcium chelating capability than citrate, which binds with calcium in a 3:2 ratio, meaning three calciumAttorney Docket No: 15969-034PC0

[0073] ions per two citrate molecules. DTPA can interact with calcium ions at 8 different places of the molecule simultaneously, which makes it very stable, and consequently the binding strength of DTPA to calcium is much higher than citrate. Second, citrate can be metabolized and consumed by platelets or other cells in the whole blood during storage period. In contrast, DTPA can preserve its chelating activity without decreasing its concentration during storage, and thus it can be used as an anticoagulant to replace citrate.

[0074] Interestingly, it is demonstrated herein that DTPA composition DPDA-1 has a better capability in preserving platelet numbers and coagulation function than citrate composition, CPDA-1. Both compositions comprise sugar source and adenine, but DPDA-1 preserved more platelets than CPDA-1 (Figure 3), and faster restoration of platelet coagulation functions in various TEG tests (Figures 4 and 5). In addition, in whole blood anticoagulated with DPDA-1, a much less calcium concentration than the calcium concentration needed in whole blood anticoagulated with CPDA-1 is required to yielded similar TEG results. Therefore, the DTPA composition, DPDA-1 comprehensively enhances the survival of platelets during storage and their functional restoration after blood transfusion with calcium.

[0075] For example, DPDA-1 shows decreased R-time and K-time and increased alpha angle representing the speed of clot formation and maximum amplitude (MA) representing the maximum clot strength achieved when compared with CPDA-1 (Figure 4). R-time indicates how long it takes for blood to coagulate, and it measures time from the coagulation cascade starting point when a clotting factor is added into the test tube to the point where clot strength reaches 2 mm amplitudes, indicating the generation of fibrin and clot propagation. K-time is the time it takes for blood clot strength to reach from 2 mm (R-time point) to 20 mm in amplitude, which reflects the contribution of fibrinogen to clot formation and indicates the speed of clot formation. These results prove that DPDA-1 is better than CPDA-1 for platelet number and function preservation.

[0076] EXAMPLES

[0077] Example 1, Materials and Methods

[0078] 1.1 ChemicalsAttorney Docket No: 15969-034PC0

[0079] The chemicals used for this study are summarized in Table 1.

[0080] Table 1. List of Reagents and Vendors

[0081] Vendor Chemical / Agent

[0082] Sigmal Aldrich Trisodium citrate

[0083] St. Louis, MO Citric acid monohydrate

[0084] Dextrose anhydrous

[0085] Adenine

[0086] Monobasic Sodiumphoshpate monohydrate

[0087] DTPA 40%

[0088] Becton-Dickinson Vacutainers

[0089] Franklin Lakes, NH

[0090] Haemonetics Corp. Disposable TEG cups

[0091] Boston, MA Ca 0.2 M

[0092] Biolegend PerCP anti-human CD42b Antibody

[0093] San Diego, CA Pacific Blue™ anti-human CD45 Antibody

[0094] Human TruStain FcX™ (Fc Receptor Blocking

[0095] Solution)

[0096] BD Sciences FETC Mouse Anti-Human PAC-1 Antibodiy

[0097] Franklin Lakes, NJ PE Mouse Anti-Human CD62P Antibodiy

[0098] Prolytix Bovine Lactadherin

[0099] Essence Junction, VT

[0100] ThermoFisher Hank’s balanced salt solution

[0101] Waltham, MA Alexa Fluor™ 647 Phalloidin

[0102] StatLab Hema-Diff #1 (Fixative), #2 (Xanthene), #3

[0103]

[0104] McKinney, TX (Thiazine)

[0105] 1.2 Preparation of Anticoagulants

[0106] To ensure equal test settings for both groups, both anticoagulant solutions used in this study were custom made in this laboratory following a formula published in the Federal Register of the Food and Drug Administration in 1978 (Table 2); CPDA-1 was made as published13, while to make an analogous DPDA-1 solution, trisodium citrate and citric acid monohydrate were substituted with sodium DTPA 40% (Sigma Aldrich, St. Louis, MO). Preliminary studies (data not shown) indicated that DTPA at a concentration of 0.12% (2.385 mmol) within the final sample yielded indefinite anticoagulation as well as maximized platelet counts. To ensure the same level of dilution within the final sample, DPDA-1 was made with a DTPA concentration of 0.977% (19.416 mM). Solutions were pH balanced to a pH of 5.9 using 6N HC1. pH was determined using a pHAttorney Docket No: 15969-034PC0

[0107] Meter (Orion Star A211 , ThermoScientific, Waltham, MA). Ingredients were weighed using a Microbalancc scale (XP26 Excellence Plus XP, Mettler Toledo, Columbus, OH).

[0108] Table 2. Formulas for CPDA-1 and DPDA-1.

[0109] CPDA-1 DPDA-1

[0110] Ingredient Concentration Ingredient Concentration (mM)

[0111] Dextrose anhydrous 169.8 Dextrose anhydrous 169.8 Adenine 2.03 Adenine 2.03 Monobasic sodium 16.09 Monobasic sodium phosphate 16.09 phosphate monohydrate

[0112] monohydrate

[0113] Trisodium citrate 89.43 Diethylenetriamine-pentaacetic 19.42 dihydrate acid pentasodium salt solution

[0114] 40%

[0115] Citric acid 15.56

[0116]

[0117] monohydrate

[0118] Ingredients were diluted in sterile water and pH balanced to a final pH of 5.9 using 6N HC1.

[0119] 1.3 Ethics Statement

[0120] Experiments within this study were carried out according to the principles of the Declaration of Helsinki. The study followed a standard operating procedure approved by the U.S. Army Institute of Surgical Research. This research was found to be IRB exempt and followed protocol H-21-012nh.

[0121] 1.4 Sample Collection

[0122] Donors were free from anti-platelet agents within 7 days prior to blood collection. After sterile venipuncture, 10 ml whole blood from healthy humans was collected into a sterile syringe without anticoagulant. The sample was thereafter aliquoted into 5 ml samples and mixed with 1.4 ml of either anticoagulant. Samples were finally transferred to a mini-blood storage bag and then stored at 4 °C for 7 days until further testing. A total of 12 samples were obtained (2 samples per donor; 1 each of DPDA-1 and CPDA-1) from 6 healthy donors (40% male, 26.33 ± 2.21 years).Attorney Docket No: 15969-034PC0

[0123] 1.5 Complete Blood Count & Histology

[0124] To evaluate the effect of either anticoagulant on red blood cell, white blood cell and platelet counts, a complete blood count was obtained using a hematology system (Advia 2120i, Siemens, Princeton, NJ) after 7 days of storage. Additionally, hemoglobin, hematocrit, and red blood cell features (mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration) were evaluated. In addition, blood smears were prepared to visually assess cell toxicity after storage. Blood smears were performed in triplicate by this institute’s Department of Comparative Pathology. After air-drying, blood smears were stained using a Giemsa / Romanowsky stain (Hema-Diff, StatLab, McKinney, TX) as a differential stain according to the manufacturer’s instructions.

[0125] 1.6 Thromboelastographic Assessment

[0126] Thromboelastographic assessment (TEG) was used to determine the clot parameters of samples anticoagulated with either anticoagulant both with and without recalcification after 7 days of storage. Assays were performed using a coagulation analyzer (Haemoscope TEG 5000, Haemonetics Corp. Boston, MA). To focus on the bare effect of the anticoagulant on the coagulability, it was decided to perform TEG assays natively without adding promoters of coagulation other than calcium. Preliminary findings indicated that a calcium concentration of 5.5 mM in whole blood anticoagulated with DPDA-1 yielded equal TEG results as a calcium concentration of 11.11 mM in whole blood treated with CPDA-1. To ensure the same level of dilution, 340 ul of whole blood from the DPDA-1 group were mixed with 10 pl of calcium 0.2 M and 10 pl of water, while 340 ul of whole blood from the CPDA-1 group were mixed with 20 pl of calcium 0.2 M. For all samples without recalcification, 340 ul of whole blood were mixed with 20 pl of water prior to TEG assessment.

[0127] 1.7 Flow Cytometry

[0128] Flow cytometry was used to assess platelet activation in whole blood after 7 days of storage. CD42 is a platelet surface marker seen in both activated and resting platelets and wasAttorney Docket No: 15969-034PC0

[0129] used to identify platelets. Procaspase-activating compound- 1 (PAC-1) antibodies were used to evaluate conformational platelet changes, while lactadhcrin was used to identify phosphatidylserine. Platelet degranulation due to activation was assessed by staining for P-selectin and CD62p. Phalloidin was used to determine platelet integrity. 5 pL of whole blood were stained with 1 pL CD42b, 2.5 pL CD62P, 1 pL bovine lactadherin (Prolytix, Essence Junction, VT), 5 pL PAC-1, and 1 pLphalloidin. Cells were stained in a total volume of 100 pL using Hank’s Balanced Salt Solution (HBSS) for 15 minutes in the dark at room temperature. Following staining, samples were resuspended in 300 pL HBSS and analyzed on a BD FACS Symphony A5 SE flow cytometer (BD Biosciences, Franklin Lakes, NJ). Forward scatter (FSC) and side scatter (SSC) parameters were used to identify platelet population based on size and shape. For this panel, 50,000 platelet FSC / SSC events were recorded.

[0130] 1.8 Sample Size Calculation

[0131] Preliminary data demonstrated a higher platelet count in DPDA-1 samples (154.75 ± 20.51 x 103cells / pl) compared with CPDA-1 samples (113.25 ± 25.05 x 103cells / pl) after brief storage. A priori power analysis indicated that 6 participants would be needed per group to detect a difference in platelet count of this size, with an a=0.05 and 1- =0.8.

[0132] A total of 12 samples were obtained (2 samples per donor) from 6 healthy donors (40% male, 26.33 ± 2.21 years).

[0133] 1.9 Statistical Analysis

[0134] Statistical analysis was carried out using GraphPad Prism 9.4.1 (GraphPad Software, San Diego, CA). Based on preliminary findings regarding increased platelet counts after storage (data not shown), an a priori power analysis suggested the enrollment of 6 donors to identify differences in platelet count after storage in CPDA- 1 or DPDA- 1. Data are presented as means ± standard deviations. The Shapiro-Wilk test was used to determine the distribution of data. Group differences after storage were found to be normal and evaluated using a paired Student’s t-test. Results with a p-value below 0.05 were considered statistically significant.Attorney Docket No: 15969-034PC0

[0135] Example 2, Complete Blood Count

[0136] While no difference between treatment groups was seen for red and white blood cells counts, platelet count was higher in DPDA-1 samples compared with CPDA-1 samples (144.0 ± 40.80 x 103cells / pl vs 118.8 ± 37.89 x 103cells / pl, p = 0.0023, Figure 3). Significant difference was also observed in MPV (mean platelet volume, indicating the average size of platelets) (CPDA-1 11.52 ± 0.58 fl vs DPDA-1 13.35 ± 0.64 fl, p = 0.0004). Additionally, MCV (mean corpuscular volume, indicating the average size of red blood cells) was slightly but significantly higher in DPDA-1 samples (CPDA-1 90.53 ± 2.04 fl vs DPDA-1 92.87 ± 2.33 fl, p = 0.0024; normal range 80-100 fl). Further cell counts and morphological parameters are included in Table 5.

[0137] Example 3. Thromboelastographic Assessment

[0138] No clotting was detected by TEG in samples from either group without calcium. However, all recalcified samples showed restored coagulability. R-Time (DPDA-1 10.90 ± 3.38 min vs CPDA-1 13.33 ± 3.02 min, p = 0.0029) and K-Time (DPDA-1 3.04 ± 1.04 min vs CPDA-1 4.64 ± 1.58 min, p = 0.0079) were shorter in samples treated with DPDA-1 compared with the CPDA-1 group, while alpha-angle was greater in the DPDA-1 group (DPDA-1 52.2 ± 8.74° vs CPDA-1 41.55 ± 11.43°, p = 0.0173, Table 3 and Figure 4).

[0139] Table 3. Viscoelastic Testing

[0140] Parameter CPDA-1 DPDA-1 p-value R (min) 13.33 + 3.02 10.90 + 3.38 0.0029

[0141] K (min) 4.64+ 1.58 3.04 + 1.04 0.0079

[0142] a (degrees) 41.55 ± 11.43 52.2 ± 8.74 0.0173

[0143] MA (mm) 54.79 + 6.23 58.90 + 4.36 0.0523

[0144]

[0145] LY30 (%) 0.00 ± 0.00 0.00 ± 0.00 n / a Abbreviations: R reaction time; K kinetics, a alpha angle; MA maximum amplitude; LY30 Lysis at 30 min

[0146] Example 4, Flow Cytometry

[0147] A higher fraction of platelets stained positively by phalloidin in DPDA-1 samples was observed compared with CPDA-1 samples (7.32 ± 2.36% vs 3.1 ± 2.36%, p = 0.0193). No other stains revealed significant differences between groups. Results are summarized in Table 4.Attorney Docket No: 15969-034PC0

[0148] Table 4. Flow Cytometry

[0149] Parameter CPDA-1 DPDA-1 p-value CD62p+ PLT [%] 20.33 ± 6.45 22.42 + 5.02 0.5064

[0150] PAC-1+ PLT [%] 5.88 ± 2.28 5.73 + 0.88 0.8977

[0151] PS+ PLT [%] 1.32 ± 1.10 0.15 ± 0.08 0.0625

[0152]

[0153] Phalloidin+ PLT [%] 3.10 + 1.16 7.32 + 2.36 0.0193 Abbreviations: PLT platelets; PAC-1 procaspase activating component- 1; PS phosphatidylserine.

[0154] Example 5. Histology

[0155] Cytological evaluation of prepared blood smears revealed no discernable difference between the CPDA-1 and DPDA-1 samples (Figure 6). The quantity and individualization of the platelets observed within the monolayer was the same regardless of which anticoagulant was utilized. Within all samples the red blood cells exhibited moderate to marked changes (echinocytes) consistent with sample aging of 7 days. Frequently throughout all samples, the neutrophils were either lysed completely, or if intact contained nuclei that were markedly swollen, with a less condensed chromatin pattern. These changes are also consistent with aging of the samples.

[0156] Table 5. Complete Blood Count

[0157] Parameter CPDA-1 PPDA-1 p-Value WBC (x 103cells / pl) 6.23 + 0.97 6.02 ± 1.18 0.2753

[0158] RBC (x 106cells / pl) 4.39 + 0.42 4.33 ±0.34 0.5611

[0159] HGB (g / dl) 13.75 ± 1.37 13.67 ± 0.83 0.8274

[0160] HCT (%) 39.73 + 3.22 40.15 ± 2.34 0.7160 MCV (11) 90.53 ± 2.04 92.87 ± 2.33 0.0024 MCH (pg) 31.28 ± 0.74 31.65 ± .79 0.0817 MCHC (g / dl) 34.55 ± 0.89 34.08 ± 0.79 0.0567

[0161] PLT (x 103cells / pl) 118.8 ± 37.89 144.0 ± 40.80 0.0023

[0162] MPV (fl) 11.52 ± 0.58 13.35 ± 0.64 0.0004

[0163]

[0164] PCT (%) 0.14 ±0.05 0.18 ±0.04 0.0009 Abbreviations: WBC white blood cell count; RBC red blood cell count, HGB hemoglobin; HCT hematocrit; MCV mean corpuscular volume; MCH mean corpuscolar hemoglobin; MCHC mean corpuscular hemoglobin concentration; PLT platelet count; MPV mean platelet volume; PCT plateletcrit.Attorney Docket No: 15969-034PC0

[0165] REFERENCES

[0166] 1. Parmeggiani A, Bowman R. Regulation of phosphofructokinase activity by citrate in normal and diabetic muscle. Biochemical and biophysical research communications 1963; 12: 268-73.

[0167] 2. Garland P, Randle P, Newsholme E. Citrate as an intermediary in the inhibition of phosphofructokinase in rat heart muscle by fatty acids, ketone bodies, pyruvate, diabetes and starvation. Nature 1963;200: 169-70.

[0168] 3. Passonneau JV, Lowry OH. The role of phosphofructokinase in metabolic regulation. Advances in enzyme regulation 1964;2: 265-74.

[0169] 4. Crochet RB, Kim JD, Lee H, Yim YS, Kim SG, Neau D, Lee YH. Crystal structure of heart 6- phosphofructo-2-kinase / fructose-2, 6-bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding. Proteins: Structure, Function, and Bioinformatics 2017;85: 117-24.

[0170] 5. Mosaoa R, Kasprzyk-Pawelec A, Fernandez HR, Avantaggiati ML. The mitochondrial citrate carrier SLC25A1 / CIC and the fundamental role of citrate in cancer, inflammation and beyond. Biomolcculcs 2021;ll: 141.

[0171] 6. Williams NC, O’Neill LA. A role for the Krebs cycle intermediate citrate in metabolic reprogramming in innate immunity and inflammation. Frontiers in immunology 2018;9: 141. 7. Infantine V, Pierri CL, lacobazzi V. Metabolic routes in inflammation: the citrate pathway and its potential as therapeutic target. Current Medicinal Chemistry 2019;26: 7104-16.

[0172] 8. Schriner J, Van Gent J, Meledeo M, Olson S, Cotton B, Cox C, Gill B. Impact of Transfused Citrate on Pathophysiology in Massive Transfusion. Critical Care Explorations 2023 ;5.

[0173] 9. Kyle T, Greaves I, Beynon A, Whittaker V, Brewer M, Smith J. Ionised calcium levels in major trauma patients who received blood en route to a military medical treatment facility. Emergency Medicine Journal 2018 ;35: 176-9.

[0174] 10. Vasudeva M, Mathew JK, Groombridge C, Tee JW, Johnny CS, Maini A, Fitzgerald MC.

[0175] Hypocalcemia in trauma patients: A systematic review. J Trauma Acute Care Surg 2021 ;90: 396- 402.

[0176] 11. Feth M, Hainline RV, Barrera G, Meledeo MA, Ross E. Pyrophosphate as a novel anticoagulant for storage of whole blood: A proof-of-concept study. Transfusion 2023.

[0177] 12. Katz AK, Glusker JP, Beebe SA, Bock CW. Calcium ion coordination: a comparison with that of beryllium, magnesium, and zinc. Journal of the American Chemical Society 1996;118: 5752-63.

[0178] 13. Additional standards for human blood and blood products, anticoagulant Cirate phosphate dextrose adenine solution. Federal Register, 1978.

[0179] 14. Braverman MA, Smith A, Pokorny D, Axtman B, Shahan CP, Barry L, Corral H, Jonas RB, Shiels M, Schaefer R. Prehospital whole blood reduces early mortality in patients with hemorrhagic shock. Transfusion 2021;61: S15-S21.

[0180] 15. Cap AP, Beckett A, Benov A, Borgman M, Chen J, Corley JB, Doughty H, Fisher A, Glassberg E, Gonzales R, Kane SF, Malloy WW, Nessen S, Perkins JG, Prat N, Quesada J, Reade M, Sailliol A, Spinella PC, Stockinger Z, Strandenes G, Taylor A, Yazer M, Bryant B, Gurney J. Whole Blood Transfusion. Military Medicine 2018;183: 44-51.

[0181] 16. Aaseth J, Nurchi VM, Andersen O. Clinical therapy of patients contaminated with polonium or plutonium. Current Medicinal Chemistry 2021 ;28: 7238-46.

[0182] 17. Fisher D, Mays CW, Taylor G. Ca—DTPA toxicity in the mouse fetus. Univ, of Utah, Salt Lake City; 1975.

Claims

Attorney Docket No: 15969-034PC0CLAIMSWhat is claimed is:

1. A composition for blood product preservation at room temperature and in refrigeration or freezing conditions, the composition comprising:diethylenetriamine pentaacetic acid (DTPA), optionally pentasodium DTPA, as an anticoagulant, a pH buffer, a sugar source, and, optionally, adenine.

2. The composition of claim 1, wherein the composition is a solution to be 5-fold to 20-fold, optionally about 10-fold diluted in a blood product suspension.

3. The composition of claim 2, wherein the concentration of DTPA is 0.5-2% w / v, optionally about 1% w / v (about 20 mM), to make a final concentration of DTPA of 0.025-0.4% w / v, optionally about 0.1% w / v (2 mM) when added to the blood product suspension.

4. The composition of any of claims 1-3, wherein the pH buffer is monobasic sodium phosphate monohydrate, and its concentration is 10-25 mM, optionally about 16 mM at pH 5.6-pH7.6, optionally about pH 5.9.

5. The composition of any of claims 1-4, wherein the sugar source is dextrose anhydrous, and its concentration is 150-200 mM, optionally about 170 mM.

6. The composition of any of claims 1-5, wherein the composition comprises adenine at a concentration of 0.5-5 mM, optionally about 2 mM.

7. The composition of any of claims 1-6, wherein the composition is optionally packed in a bag or container selected from syringe, tube, vial or ampule for blood product collection.

8. The composition of any of claims 2-7, wherein the blood product comprises whole blood, red blood cells (RBCs), platelets, or combination thereof, and wherein (i) when the blood product comprises RBCs, the RBCs are suspended in a red blood cell preservation solution comprising adenine, dextrose, sorbitol, sodium chloride, and mannitol, and (ii) when the blood product comprises platelets, the platelets are suspended in plasma, a platelet additive solutions (PAS), or other physiological salt solution optionally selected from Frog’s Ringer’s solution, mammalian Ringer’s solution, Ringer’s lactate solution, Krebs Henseleit salt solution, Tyrode solution, Ringer locke solution, and De Jalon solution; and packed in a bag or a container with a volume capacity of 1-1000 mL.Attorney Docket No: 15969-034PC09. The composition of claim 8, wherein the blood product is platelets.

10. A method for preserving platelets, the method comprising:adding an effective amount of the composition of any of claims 1-9, in solution form, to a platelet suspension to form a suspension / composition mixture; andstoring the suspcnsion / mixturc at room temperature, or refrigerating or freezing the platelet suspension with the composition at a temperature of 4°C or below,wherein the volume of the solution comprising the composition to be added to the platelet suspension is for 5-fold to 20-fold, optionally 10-fold dilution in the final suspension / mixture volume.

11. A bag or container for the collection of whole blood, RBC, or platelet, the bag comprising an amount of the composition of any of claims 1-9.

12. The bag or container of claim 11, wherein, the volume of the solution comprising the composition is less than 20%, optionally less than 10% of the total volume of the bag or container.

13. Blood collection double / triple / quadruple / quintuple bag system, wherein the main bag and platelet collection bag comprise a solution comprising the composition of any of claims 1-9.

14. A test tube, in which DTPA anticoagulant is coated on the internal surface of the tube.

15. A regional anticoagulation solution for use in procedures that require regional anticoagulation, such as in CVVHDF.

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