Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

TITLE OF THE INVENTION
ANTICOAGULANT COMPOSITION AND METHODS THEREOF

APPLICANT:
Meril Medical Innovations Private Limited, an Indian company of the address Survey No 1574 (Old No 135/139), Bilakhia House, Muktanand Marg, Chala, Vapi, Valsad, Gujarat, 396191 India.

The following specification particularly describes the invention and the manner in which it is to be performed:
 
FIELD OF INVENTION
[001] The present disclosure relates to an anticoagulant composition and methods thereof. In particular, the present disclosure relates to a citrate-based anticoagulant composition and methods thereof.
BACKGROUND OF THE INVENTION
[002] Coagulation of blood is usually a natural response to a bleeding trauma. The bleeding trauma may occur, for example, when a blood sample is being drawn from an individual for diagnostic and/or therapeutic purposes. The clotting of blood is influenced by a plurality of blood clotting factors present in the blood itself that regulates a natural coagulation cascade. However, from a medical stand point, once the blood sample drawn from an individual coagulates, the blood sample loses its diagnostic and therapeutic significance. A few exemplary diagnostic and therapeutic use of blood sample includes apheresis, blood donation, laboratory testing, etc.
[003] To prevent or slow down coagulation of blood, an anticoagulant (or blood thinner) is added to the blood sample before it is processed for diagnostic or therapeutic purposes. The anticoagulant binds with and blocks the receptors of blood clotting factors, thereby disrupting the natural coagulation cascade.
[004] One prominent use of the blood sample drawn from individuals is preparation of platelet rich plasma (PRP) for hair treatment, skin rejuvenation, wound healing, synthetic bone graft, etc. To achieve significant therapeutic efficacy from PRP-based treatments, the PRP prepared should have very high concentration of platelets (i.e., good recovery rate), structural integrity of the constituents of the PRP should be maintained for long duration, the pH of the PRP should be regulated and maintained close to the physiological pH of blood.
[005] Conventionally, ethylenediaminetetraacetic acid (EDTA) or salts thereof, are used as general-purpose anticoagulants for the preparation of PRP from blood sample.
[006] However, the conventionally used general-purpose anticoagulants fail to provide desired quality PRP from blood sample. For instance, either the platelet recovery rate is poor to be of any use, or the structural integrity of the constituents of the PRP is destroyed. More often than not, the PRP prepared using the conventional anticoagulants does not have pH close to the physiological pH of the blood, resulting in physical discomfort once the said PRP is used as a therapeutic agent against an individual.
[007] Another common disadvantage associated with the conventional anticoagulants is that they fail to provide long shelf life to the blood sample drawn from individuals. In other words, the lymphocytic vigor of the blood sample mixed with conventional anticoagulant is lost after a few hours, say, 4-6 hours. Since, quite a few phlebotomists draw blood sample far away from blood testing centers, the blood sample are required to be transported to the blood testing centers for long periods of time. During this time, the loss in lymphocytic vigor introduces undesirable error in the medical reports when the blood sample are tested in the blood testing centers.
[008] Therefore, there arises a need for an anticoagulant composition that overcomes the problems associated with the conventional anticoagulant compositions.
SUMMARY OF INVENTION
[009] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings, however, it is to be understood that the disclosed embodiments are mere examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.
[0010] In an exemplary embodiment, the present disclosure relates to a composition including at least one anticoagulating agent, at least one pH regulator, at least one osmolyte, dissolved in a pre-defined amount of an aqueous solvent. The anticoagulating agent is present in the concentration ranging from 0.5 g/L to 50 g/L. The pH regulator is present in the concentration ranging from 0.1 g/L to 20 g/L. The osmolyte is present in the concentration ranging from 5 g/L to 100 g/L. The pH of the composition ranges from 4.5 to 5.5.
[0011] In another exemplary embodiment, the present disclosure relates to a method to prepare a composition. the method commences by adding at least one anticoagulant agent, at least one pH regulator, and at least one osmolyte in respective pre-defined concentration to an aqueous solvent to obtain a mixture. The pre-defined concentration of the at least one anticoagulating agent ranges between 0.5 g/L to 50 g/L. The pre-defined concentration of the at least one pH regulator ranges between 0.1 g/L to 20 g/L. The pre-defined concentration of the at least one osmolyte ranges between 5 g/L to 100 g/L. The mixture obtained is subjected to a mixing technique for a pre-defined amount of time period to obtain a solution. The pH of the composition is checked. The composition is sterilized provided the pH of the composition ranges from 4.5 to 5.5.
[0012] In yet another exemplary embodiment, the present disclosure relates to a method to prepare platelet rich plasma (PRP). The method commences by adding a pre-defined amount of a composition as described above per unit volume of a blood sample to obtain a treated blood sample. The composition ranging from 150 µL/mL of the blood sample to 250 µL/mL of the blood sample. The treated blood sample obtained is subjected to centrifugation at a pre-defined speed for a pre-defined time period to obtain a top layer and a bottom layer. The upper layer obtained is subjected to centrifugation at a pre-defined speed for a pre-defined time period to obtain a supernatant and a pellet. A portion of the supernatant obtained is discarded. The pellet obtained is resuspended in the remaining supernatant to obtain the platelet rich plasma.
BRIEF DESCRIPTION OF DRAWINGS
[0013] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the apportioned drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentality disclosed herein. Moreover, those in the art will understand that the drawings are not to scale.
[0014] Fig. 1 depicts a method 100 to prepare a composition, according to an embodiment of the present disclosure.
[0015] Fig. 2 depicts a method 200 to prepare platelet rich plasma (PRP) using the composition, according to an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE DRAWINGS
[0016] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and/or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, coupled to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have a property of, or the like. Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.
[0017] Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean “one or more but not all embodiments” unless expressly specified otherwise. The terms “including,” “comprising,” “having,” and variations thereof mean “including but not limited to” unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and/or mutually inclusive, unless expressly specified otherwise. The terms “a,” “an,” and “the” also refer to “one or more” unless expressly specified otherwise.
[0018] Although the method steps of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of method steps other than the particular, sequential order disclosed. For example, method steps described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.
[0019] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.
[0020] The present disclosure relates to an anticoagulant composition (or composition) and methods of preparation thereof. The present disclosure further relates to a method to prepare platelet rich plasma using the composition. The composition prevents and/or slows down coagulation (or clotting) of blood in a blood sample for erythrocyte survival/preservation and blood storage. The composition may be used for isolation/preparation of platelet rich plasma (PRP) from a blood sample with very high recovery rate of platelets. The composition of the present disclosure reduces any potential adverse reactions, ensuring safety of the individual being administered PRP obtained from the blood sample treated with the composition. The composition prolongs the shelf life of the blood sample (and the PRP derived therefrom) by maintaining pH and preventing coagulation for long periods of time.
[0021] The composition of the present disclosure includes a pre-defined amount of at least one anticoagulating agent, a pre-defined amount of at least one pH regulator, and a pre-defined amount of at least one osmolyte dissolved in a pre-defined amount of an aqueous solvent.
[0022] The anticoagulating agent is at least one of trisodium citrate (also known as sodium citrate) having formula I, trisodium citrate dehydrate, dipotassium citrate, ethylenediaminetetraacetic acid (EDTA), heparin sulfate, etc. The concentration (amount) of the anticoagulating agent present in the composition, ranges from 0.5 g/L to 50 g/L. In an exemplary embodiment, the anticoagulating agent includes trisodium citrate dehydrate. In an exemplary embodiment, the amount of trisodium citrate dehydrate present is 25 g/L. The anticoagulating agent prevents coagulation of the blood sample by binding with the ionized calcium ions present in the blood sample, thereby forming a non-ionized calcium citrate. Since, calcium ions are one of the critical stakeholders of the natural coagulation cascade, reducing the amount of ionized calcium ions in the blood prevents and/or slows down the coagulation of the blood sample.
(Formula I)
[0023] The pH regulator is at least one of citric acid having formula II, acetic acid, phosphoric acid, lactic acid, sodium bicarbonate, hydrochloric acid, sodium hydroxide, phosphate buffers, etc. The concentration (amount) of the pH regulator present in the composition, ranges from 0.1 g/L to 20 g/L. In an exemplary embodiment, the pH regulator includes citric acid. In an exemplary embodiment, the amount of citric acid present is 10 g/L. The pH regulator provides an acidifying effect and lowers the pH of the blood sample to about 6.5. The lower pH maximizes platelet recovery during PRP preparation and impairs the activation of any residual thrombin (one of the blood clotting factors). The pH regulator helps in maintaining the pH of the blood sample (and the PRP derived therefrom) close to the physiological pH of the blood for prolonged period of time, for example, during storage and transportation.
(Formula II)
[0024] The osmolyte is at least one of dextrose monohydrate (also called hydrated D-glucose) having formula III, sorbitol, sucrose, mannitol, glycerol, trehalose, etc. The concentration (amount) of the osmolyte present in the composition, ranges from 5 g/L to 100 g/L. In an exemplary embodiment, the osmolyte includes dextrose monohydrate. In an exemplary embodiment, the amount of dextrose monohydrate present is 35 g/L. The monohydrate corresponds to the water of crystallization associated with the dextrose molecule. The water of crystallization helps to prevent quick absorption inside the body resulting in less chance of irritation or other undesirable effects.
(Formula III)
[0025] The osmolyte maintains isotonicity of the constituents (for example, erythrocytes, platelets, plasma, etc.) of the blood sample (and the PRP prepared therefrom). In other words, the osmolyte prevents a plurality of cells present in the blood sample to either shrink or swell, thereby maintaining structural integrity (and lymphocytic vigor) of the plurality of cells in the blood sample for long periods of time. In an exemplary embodiment, the osmolyte preserves the lymphocytic vigor of the blood sample for 48 hours to 72 hours.
[0026] The aqueous solvent is at least one of water, ultra-pure water, buffered saline, phosphate-buffered solution, etc. The amount of aqueous solvent in the composition depends upon the amount of the anticoagulating agent, the pH regulator, and the osmolyte as described above. In an exemplary embodiment, the aqueous solvent is ultra-pure water. The aqueous solvent behaves as a solvent in which the anticoagulating agent, the pH regulator, and the osmolyte is dissolved for convenient addition to blood sample as required.
[0027] Additionally or optionally, the aqueous solvent is sterilized using, for example, an autoclave or a 0.22 micron filter. The sterility of the aqueous solvent ensures the blood sample is not contaminated or infected by the composition of the present disclosure.
[0028] Now referring the figures, Fig. 1 depicts an exemplary method 100 for preparing the composition of the present disclosure. The method 100 commences at an optional step 101 by sterilizing the aqueous solvent. The aqueous solvent may be sterilized using at least one of an autoclave and filtration technique, etc. The aqueous solvent is at least one of water, ultra-pure water, buffered saline, phosphate-buffered solution, etc. In an exemplary embodiment, the aqueous solvent is ultra-pure water. The ultra-pure water is sterilized by autoclaving at 121 °C, 15 psi for 30 mins. Alternatively, the ultra-pure water is filtered using a 0.22 micron filter (made of polyether sulfone (PES) membrane).
[0029] At step 103, the pre-defined amount of anticoagulant agent, the pre-defined amount of the pH regulator, and the pre-defined amount of the osmolyte are added to the pre-defined amount of the aqueous solvent to obtain a mixture. The anticoagulating agent is at least one of trisodium citrate (also known as sodium citrate) having formula I, trisodium citrate dehydrate, dipotassium citrate, ethylenediaminetetraacetic acid (EDTA), heparin sulfate, etc. The amount of anticoagulating agent in the composition ranges from 0.5 g/L to 50 g/L.
[0030] The pH regulator is at least one of citric acid having formula II, acetic acid, phosphoric acid, lactic acid, sodium bicarbonate, hydrochloric acid, sodium hydroxide, phosphate buffers, etc. The amount of pH regulator in the composition ranges from 0.1 g/L to 20 g/L.
[0031] The osmolyte is at least one of dextrose monohydrate (also called hydrated D-glucose) having formula III, sorbitol, sucrose, mannitol, glycerol, trehalose, etc. The amount of osmolyte in the composition ranges from 5 g/L to 100 g/L.
[0032] In an exemplary embodiment, 25 g of trisodium citrate dehydrate, 10 g of citric acid and 35 g of dextrose monohydrate is added to 1 L of ultra-pure water to obtain the mixture.
[0033] At step 105, the mixture obtained from step 103 is subjected to a mixing technique for a pre-defined amount of time period to obtain a solution. The mixing technique is at least one of mechanical shaking, mechanical stirring, magnetic stirring, ultrasonication, vortex mixing, high-speed homogenization, etc. The pre-defined amount of time period ranges from 5 min to 60 min. In an exemplary embodiment, the mixture is homogenously mixed with the help of a magnetic stirred until all of the anticoagulant agent, the pH regulator and the osmolyte are completely dissolved in the aqueous solvent.
[0034] At step 105a, the pH of the solution obtained from step 105 is checked using a pH meter. The pH of the solution (and the composition) ranges from 4.5 to 5.5. In an exemplary embodiment, the pH of the solution (and the composition) is 4.49. The conductivity of the solution is measured using a conductivity meter. The conductivity of the composition ranges from 11 mS/cm to 14 mS/cm. In an exemplary embodiment, the conductivity of the composition is 8.2 mS/cm. The conductivity ensures proper ionic strength of the first composition for further applications.
[0035] If the pH of the solution obtained from the step 105 is between 4.5 and 5.5, the solution obtained from step 105 is the composition.
[0036] At an optional step 107, if the pH of the solution obtained from step 105 is not between 4.5 to 5.5, the pH of the solution obtained from the step 105 is adjusted to obtain the composition.
[0037] If the pH of the solution is less than the pH of the composition, a pre-defined amount of a base (or alkali) is added to the composition. If the pH of the solution is more than the pH of the composition, a pre-defined amount of an acid is added to the solution. The amount of acid or base added to the solution depends upon the difference in pH of the solution and the pH of the composition.
[0038] In an exemplary embodiment, the pH of the solution is reduced by dropwise adding the acid while monitoring the pH of the solution using a pH meter (Pico+ pH meter, procured from Labindia Analytical). In another exemplary embodiment, the pH of the solution is increased by dropwise adding the base while monitoring the pH of the solution using a pH meter (Pico+ pH meter, procured from Labindia Analytical).
[0039] After step 107, the step 105a is repeated to check the pH of the solution. If the pH is not between 4.5 and 5.5, step 107 is repeated until and unless the pH of the solution is between 4.5 and 5.5.
[0040] At an optional step 109, the composition obtained from step 105a having pH between 4.5 and 5.5 is sterilized by filtering the composition using at least one of one or more filters, or a combination filter. The filter is made of one or more pre-defined materials and have a pre-defined pore size. The pre-defined material may be at least one of cellulose filter, polyether sulfone (PES), modified polyether sulfone (mPES) hydrophilic membrane filter, hydrophilic polyvinylidene fluoride (PVDF), etc. The pore size of the filter ranges from 0.8 µm to 0.2 µm. In an exemplary embodiment, the composition is sterilized by passing the composition through the filter is made of PES having pore size of 0.2 µm.
[0041] The combination filter may include at least one pre-filter and at least one core-filter, each having a pre-defined pore size. The pore size of the of the pre-filter is at least one of 1.2 µm, 0.8 µm, and 0.6 µm. The pore size of the of the core-filter is less than the pore size of the pre-filter. The pore size of the core-filter is at least one of 0.45 µm, and 0.2 µm. In an exemplary embodiment, the combination filter includes one pre-filter with pore size of 0.8 µm and one core-filter with pore size of 0.2 µm. The pre-filter(s) and core-filter(s) of the combination filter are made of one or more pre-defined materials including, but not limited to, cellulose filter, polyether sulfone (PES), modified polyether sulfone (mPES) hydrophilic membrane filter, hydrophilic polyvinylidene fluoride (PVDF), etc.
[0042] In an exemplary embodiment, the composition is first passed through the pre-filter and then through the core-filter made of PES to sterilize the composition. Sterilizing the composition ensures the blood sample is not contaminated or infected by the composition of the present disclosure, thereby maintaining blood sample purity and reliability for accurate analysis/tests.
[0043] The composition obtained from method 100 may be stored at a pre-defined temperature ranging between 15 °C and 25 °C without any loss in functional activity for long periods of time.
[0044] To arrest coagulation of a blood sample, a pre-defined amount of the composition is added per unit of the blood sample. The pre-defined amount of composition added to the blood sample ranges from 150 µL/mL of blood sample to 250 µL/mL of the blood sample. In an exemplary embodiment, 250 µL of the composition is added per 1 ml of the blood sample. For preparation of PRP by adding the composition of the present disclosure with the blood sample, the recovery rate of platelets in the PRP (compared to platelets in the blood sample) is more than 85%.
[0045] Fig. 2 depicts an exemplary (ex vivo) method 200 of preparing PRP from a blood sample using the composition of the present disclosure. In other words, the composition of the present disclosure is used to separate and concentrate the platelets in plasma from the blood sample. The method 200 commences at step 201 by adding a pre-defined amount of the composition per unit of the blood sample to obtain a treated blood sample. The pre-defined amount of composition added to the blood sample ranges from 150 µL/mL of the blood sample to 250 µL/mL of the blood sample. In an exemplary embodiment, 250 µL of the composition is added to 1 ml of the blood sample to obtain the treated blood sample.
[0046] At step 203, the treated blood sample obtained from step 201 is subjected to centrifugation at a pre-defined speed for a pre-defined time period to obtain a top layer and a bottom layer. The upper layer and the bottom layer are formed (for example, in a first centrifuge tube) due to the centrifugal force applied on the treated blood sample. The predefined speed ranges from 98 g to392 g. The pre-defined time period ranges from 10 minute to 12 minute. In an exemplary embodiment, the treated blood sample is centrifuged at 98 g, room temperature for 10 min. The speed of the centrifuge is a crucial factor to preserve the plurality of cells from shear stress and also to have effective separation in the centrifuge.
[0047] At step 205, the upper layer obtained from step 203 is carefully aspirated out to a second centrifuge tube and the bottom layer is discarded. In an exemplary embodiment, the upper layer includes the platelets, plasma, white blood cells (leukocytes), and extracellular vesicles. In an exemplary embodiment, the bottom layer includes red blood cells (erythrocytes), residual leukocytes, and other cellular debris. In an exemplary embodiment, for every 1 mL of the blood sample, 500 µL of the top layer is aspirated out to the second centrifuge tube.
[0048] At step 207, the upper layer obtained from step 205 is subjected to centrifugation at a pre-defined speed for a pre-defined time period to obtain a supernatant (or platelet poor plasma, PPP) and a pellet. The supernatant and the pellet are formed (for example, in the second centrifuge tube) due to the centrifugal force applied on the upper layer. The pre-defined time period ranges from 10 minute to 12 minute. In an exemplary embodiment, the treated blood sample is centrifuged at 392 g, room temperature for 10 min.
[0049] At step 209, a portion of the supernatant obtained from step 207 is discarded from the top of the second centrifuge tube. In an exemplary embodiment, 250 µL of the supernatant from the top of the second centrifuge tube is discarded.
[0050] At step 211, after discarding the portion of the supernatant, the pellet is resuspended in a remaining portion of the supernatant in the second centrifuge tube to obtain the PRP. In an exemplary embodiment, the pellet is resuspended in 250 µL of the remaining portion of the supernatant. In an exemplary embodiment, the recovery rate of platelets in the PRP (compared to platelets in the blood sample) is more than 85%.
[0051] The present disclosure will now be explained with the help of the following examples:
[0052] Example 1: Preparation of the anticoagulant composition of the present disclosure.
[0053] 1 L of ultra-pure water (Water for Injection grade) was sterilized by autoclaving at 121 °C, 15 psi for 30 mins. 25 g of sodium citrate, 10 g of citric acid, and 35 g of dextrose monohydrate was dissolved in 1 L of the ultra-pure water and mixed homogenously using a magnetic stirrer. The composition was then sterilized by passing through a 0.22 µm filter. The pH of the composition obtained was 4.49, and the conductivity was 8.2 millisiemens per centimeter (mS/cm).
[0054] Few drops of the composition were added to culture plates filled with sterilized Luria Agar and incubated at 37 °C for 24 hours. No bacterial growth was observed, thus, confirming that the composition prepared was sterile and ready for diagnostic or therapeutic use.
[0055] The composition was stored at 25 °C, 4 °C, 50 °C and 37 °C without any loss in functionality after 2 years.
[0056] Example 2: Platelet recovery using the composition
[0057] Step 1: 250 µL of the composition obtained from example 1 was added to 1000 µL of the blood sample and mixed homogenously. The blood sample was tested for different constituents (as shown in Table 1 below) using an automated hematology analyzer (KX-21, procured from Sysmex).
[0058] Step 2: the blood sample was centrifuged at 98 g for 10 mins. 400 µL of the top layer (or platelet poor plasma, PPP) was aspirated out and tested for different constituents (as shown in Table 1 below) using the automated hematology analyzer (KX-21, procured from Sysmex).
[0059] Step 3: 400 µL of the top layer was centrifuged at 392 g for 10 mins. 200 µL of the supernatant was discarded from the top and the pellet was resuspended in the remaining supernatant to obtain platelet rich plasma (PRP). The PRP was tested for different constituents (as shown in Table 1 below) using the automated hematology analyzer (KX-21, procured from Sysmex). The pH was measured using a pH meter (Pico+ pH meter, procured from Labindia Analytical).
Step 1: Blood sample Step 2: platelet poor plasma (PPP) Step 3: platelet rich plasma (PRP)
Total platelet (T-PLT) 259 x 103 per µL 421 x 103 per µL 845x 103 per µL
Total platelet after 12Hrs (T-PLT12) 248 x 103 per µL 421 x 103 per µL 840x 103 per µL
Mean platelet volume (MPV) 10.5 fl 8.5 fl 8.4 fl
Platelet distribution width (PDW) 15.1 fl 10.5 fl 10.4 fl
Red blood cell (RBC) 4.86 x 106 per µL 0.05 x 106 per µL 0.04 x 106 per µL
Red blood cell after 12 hours (RBC12) 4.60 x 106 per µL 0.02 x 106 per µL 0.01 x 106 per µL
White blood cell (WBC) 3.7 x 103 per µL 0.4 x 103 per µL 0.3 x 103 per µL
White blood cell after 12 hours (WBC12) 3.6 x 103 per µL 0.3 x 103 per µL 0.3 x 103 per µL
Plasma volume 1000 µL 600 µL 300 µL
pH 6.5 6.2 6.4
Platelet recovery rate (compared to blood sample) - 97.5% 97.8%
(Table 1)
[0060] It was evident from the above table that the composition of the present disclosure provided 97.8% platelet recovery compared to the blood sample. Further, the pH was maintained close to the pH of the physiological pH of the blood. Even after 12 hours, the structural integrity of the blood cells were maintained.
[0061] Example 3: Platelet recovery with different composition and different pH
[0062] Different compositions were prepared, each having different pH using the same method as outlined in Example 1 above. The compositions prepared were then used to test platelet recovery (of PRP compared to platelets in the blood sample) using the same method as outlined in Example 2 above. The compositions with respective pH and platelet recovery are tabulated in table 2 below. The last row of table 2 represents the composition of the present disclosure.
pH of the composition Composition Platelet recovery rate of PRP (%)
Sodium citrate (g) Citric Acid (g) Dextrose monohydrate (g) Phosphate Buffer saline (mm) Water (mL)
5.2 0.31 0.073 0.269 - 10 6.7
5.3 0.323 0.076 0.277 - 10 10
5.1 0.352 0.09606 0.277 - 10 10
5.3 0.294 0.057 0.237 - 10 8.4
5.17 0.441 0.096 0.297 - 10 7.7
5.36 0.588 0.096 0.297 - 10 11.8
5.36 0.441 0.076 0.396 - 10 20
5.52 0.588 0.076 0.396 - 10 17
4.49 0.352 0.192 0.277 - 10 22
4.49 0.235 0.288 0.198 - 10 27
4.49 0.352 0.288 0.277 - 10 20
4.49 0.033 0.012 0.036 - 10 3
4.49 0.352 0.384 0.277 - 10 48
4.49 0.235 0.384 0.198 - 10 51
4.49 0.176 0.192 0.138 - 10 33
4.49 0.349 0.48 0.277 - 10 45
4.49 0.294 0.48 0.297 - 10 30
4.49 0.235 0.432 0.198 - 10 13
4.49 0.147 0.432 0.237 - 10 12
4.49 0.352 0.96 0.277 - 10 16
4.49 0.188 0.432 0.445 - 10 29
4.49 0.235 0.384 0.198 1 10 51
4.49 0.352 0.384 0.277 2 10 39
4.49 0.235 0.384 0.198 5 (5mL) 10 29
4.49 0.0882 0.09606 0.07948 2 (2mL) 10 23
4.49 0.0882 0.09606 0.07948 - 10 28
4.49 0.25 0.1 0.35 - 10 87
(Table 2)
[0063] It is evident from the above table that the composition of the present disclosure provided the maximum platelet recovery.
[0064] Example 4: Platelet recovery of the composition of the present disclosure compared with conventionally available anticoagulants
[0065] The composition as obtained from Example 1 above was used to test platelet recovery using the method outlined in Example 2 above. The platelet recovery of the composition of Example 1 was compared with platelet recovery tested with conventionally available anticoagulants by following the same method as outlined in Example 2 above. The observations are tabulated in table 3 below. The platelet recovery % disclosed in the table 3 below is with respect to the platelets in the blood sample.
Composition of Example 1 Parameters Blood sample Platelet poor plasma (PPP) Platelet rich plasma (PRP)
pH 7.3 7.1 7.0
White blood cell (WBC) (x 103 per µL) 3.7 0.4 0.4
Red blood cells (RBC) (x 106 per µL) 5.8 0.02 0.02
Hemoglobin (HGB) (g/dL) 11.2 0.0 (-)0.1
Platelet (PLT) (x 103 per µL) 259 343 820
Plasma volume (µL) 1000 600 300
Total platelet (T-PLT) 259000000 205800000 246000000
Platelet recovery rate (%) - 79.46 94.98
Total platelet after 12 hours (T-PLT12) (x 103 per µL) 250 820
SD1 (Conventional anticoagulant) pH 7.3 7.0 7.0
White blood cell (WBC) (x 103 per µL) 3.4 0.2 0.2
Red blood cells (RBC) (x 106 per µL) 4.86 0.05 0.05
Hemoglobin (HGB) (g/dL) 10.3 (-)0.1 (-)0.1
Platelet (PLT) (x 103 per µL) 258 256 258
Plasma volume (µL) 1000 600 300
Total platelet (T-PLT) 258000000 153600000 77400000
Platelet recovery rate (%) - 59.53 50.39
Total platelet after 12 hours (T-PLT12) (x 103 per µL) 257 256
SD2 (Conventional anticoagulant) pH 7.3 7.01 7.1
White blood cell (WBC) (x 103 per µL) 3.7 0.2 0.3
Red blood cells (RBC) (x 106 per µL) 5.31 0.02 0.03
Hemoglobin (HGB) (g/dL) 11.3 0.0 (-)0.1
Platelet (PLT) (x 103 per µL) 268 220 149
Plasma volume (µL) 1000 600 300
Total platelet (T-PLT) 268000000 132000000 44700000
Platelet recovery rate (%) - 49.25 16.67
Total platelet after 12 hours (T-PLT12) (x 103 per µL) 260 148
SD3 (Conventional anticoagulant) pH 7.3 7.1 6.9
White blood cell (WBC) (x 103 per µL) 3.7 0.4 0.4
Red blood cells (RBC) (x 106 per µL) 5.07 0.01 0.01
Hemoglobin (HGB) (g/dL) 10.9 0.0 0.0
Platelet (PLT) (x 103 per µL) 256 134 90
Plasma volume (µL) 1000 600 300
Total platelet (T-PLT) 265000000 80400000 90000000
Platelet recovery rate (%) - 30.33 30.96
Total platelet after 12 hours (T-PLT12) (x 103 per µL) 258 92
SD4 (Conventional anticoagulant) pH 7.3 7.0 7.1
White blood cell (WBC) (x 103 per µL) 3.6 0.2 0.3
Red blood cells (RBC) (x 106 per µL) 5.02 0.02 0.02
Hemoglobin (HGB) (g/dL) 10.8 0.0 (-)0.1
Platelet (PLT) (x 103 per µL) 267 180 128
Plasma volume (µL) 1000 600 300
Total platelet (T-PLT) 267000000 108000000 38400000
Platelet recovery rate (%) - 40.44 14.38
Total platelet after 12 hours (T-PLT12) (x 103 per µL) 268 125
(Table 3)
[0066] It is evident from the above table that compared to the conventional anticoagulants, the composition of the present disclosure provided the maximum platelet recovery and maintained the pH close to the physiological pH of the blood. Further, the composition maintained the integrity of the blood cells even after 12 hours.
[0067] The scope of the invention is only limited by the appended patent claims. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. , Claims:WE CLAIM
1. A composition, comprising:
a. at least one anticoagulating agent present in the concentration ranging from 0.5 g/L to 50 g/L;
b. at least one pH regulator present in the concentration ranging from 0.1 g/L to 20 g/L;
c. at least one osmolyte present in the concentration ranging from 5 g/L to 100 g/L; and
d. a pre-defined amount of an aqueous solvent, the at least one anticoagulating agent, the at least one pH regulator, and the at least one osmolyte are dissolved in the aqueous solvent;
wherein, the pH of the composition ranges from 4.5 to 5.5.
2. The composition as claimed in claim 1, wherein the conductivity of the composition ranges from 11 mS/cm to 14 mS/cm.
3. The composition as claimed in claim 1, wherein the at least one anticoagulating agent includes at least one of trisodium citrate having formula I, trisodium citrate dehydrate, dipotassium citrate, ethylenediaminetetraacetic acid (EDTA), and heparin sulfate.
4. The composition as claimed in claim 1, wherein the at least one pH regulator includes at least one of citric acid having formula II, acetic acid, phosphoric acid, lactic acid, sodium bicarbonate, hydrochloric acid, sodium hydroxide, and phosphate buffers.
5. The composition as claimed in claim 1, wherein the at least one osmolyte includes at least one of dextrose monohydrate having formula III, sorbitol, sucrose, mannitol, glycerol, and trehalose.
6. The composition as claimed in claim 1, wherein the aqueous solvent is at least one of water, ultra-pure water, buffered saline, phosphate-buffered solution.
7. The composition as claimed in claim 1, wherein the composition includes 25 g of trisodium citrate dehydrate, 10 g of citric acid and 35 g of dextrose monohydrate dissolved in 1 L of ultra-pure water.
8. A method (100) to prepare a composition, the method (100) comprising:
a. adding at least one anticoagulant agent, at least one pH regulator, and at least one osmolyte in respective pre-defined concentration to an aqueous solvent to obtain a mixture, the pre-defined concentration of the at least one anticoagulating agent ranges between 0.5 g/L to 50 g/L, the pre-defined concentration of the at least one pH regulator ranges between 0.1 g/L to 20 g/L, and the pre-defined concentration of the at least one osmolyte ranges between 5 g/L to 100 g/L;
b. subjecting the mixture obtained from step a to a mixing technique for a pre-defined amount of time period to obtain the composition;
c. checking pH of the composition; and
d. sterilizing the composition provided the pH of the composition ranges from 4.5 to 5.5.
9. The method (100) as claimed in claim 8, wherein the step a. of the method (100) includes adding at least one of trisodium citrate having formula I, trisodium citrate dehydrate, dipotassium citrate, ethylenediaminetetraacetic acid (EDTA), and heparin sulfate to an aqueous solvent.
10. The method (100) as claimed in claim 8, wherein the step a. of the method (100) includes adding at least one of citric acid having formula II, acetic acid, phosphoric acid, lactic acid, sodium bicarbonate, hydrochloric acid, sodium hydroxide, and phosphate buffers to an aqueous solvent.
11. The method (100) as claimed in claim 8, wherein the step a. of the method (100) includes adding at least one of dextrose monohydrate having formula III, sorbitol, sucrose, mannitol, glycerol, and trehalose to an aqueous solvent.
12. The method (100) as claimed in claim 8, wherein before the step a. of the method (100), the method (100) includes sterilizing at least one of water, ultra-pure water, buffered saline, and phosphate-buffered solution.
13. The method (100) as claimed in claim 8, wherein the step b. of the method (100) includes subjecting the mixture to at least one of mechanical shaking, mechanical stirring, magnetic stirring, ultrasonication, vortex mixing, and high-speed homogenization.
14. The method (100) as claimed in claim 8, wherein the step d. of the method (100) includes filtering the composition using at least one of one or more filters, or a combination filter.
15. A method (200) to prepare platelet rich plasma (PRP), the method (200) comprising:
a. adding a pre-defined amount of a composition as claimed in any of the claims 1-7 per unit volume of a blood sample to obtain a treated blood sample, the composition ranging from 150 µL/mL of the blood sample to 250 µL/mL of the blood sample;
b. subjecting the treated blood sample obtained from step a. to centrifugation at a pre-defined speed for a pre-defined time period to obtain a top layer and a bottom layer;
c. subjecting the upper layer obtained from step b. to centrifugation at a pre-defined speed for a pre-defined time period to obtain a supernatant and a pellet; and
d. discarding a portion of the supernatant obtained from step c. and resuspending the pellet obtained from step c. in the remaining supernatant to obtain the platelet rich plasma.
16. The method (200) as claimed in claim 15, wherein the step b. of the method (200) includes centrifuging the treated blood sample at 98 g to 392 g for 10 mins to 12 mins.
17. The method (200) as claimed in claim 15, wherein the step c. of the method (200) includes centrifuging the upper layer at 392 g for 10 mins to 12 mins.
18. The method (200) as claimed in claim 15, wherein after step d. of the method (200) the recovery rate of platelets in the PRP compared to platelets in the blood sample is more than 85%.