DE202015009321U1 - Compositions for separating platelet-rich plasma - Google Patents

Abstract

A sample collection tube for separating a PRP fraction of whole blood comprising: a lumen tube; a polymerizable composition disposed in a lumen of the tube, the polymerizable composition comprising an oligomer, a photoinitiator, and a stabilizer; wherein the polymerizable composition is flowable with the whole blood, has a density between an average density of a PRP fraction and an average density of a non-PRP fraction, and a first average molecular weight; wherein the polymerizable composition is formulated such that it can be centrifuged with the tube to settle between the PRP fraction and a non-PRP fraction without substantial activation of platelets in the PRP fraction, wherein substantial activation based on the Ability of the platelets in the PRP fraction to aggregate to at least one of ristocetin and collagen is measurable.

Description

Subject of the invention

The subject of the invention are separation technologies.

background

The background description includes information that may be useful in understanding the present invention. It is not an admission that the information provided herein is prior art or relevant to the presently claimed invention, or that any publication that is specifically or implicitly referenced is prior art.

Platelet-rich plasma (PRP) therapy is used for bone repair and regeneration, plastic surgery, oral surgery, and eye care, and is receiving increasing recognition and recognition for its efficacy in the treatment of sports injuries, nerve injuries, tendonitis, and osteoarthritis. PRP can be considered as a concentrated source of platelets that can be delivered to the site of injury and activated to allow rapid release of the growth factor and stimulation of bone or soft tissue recovery. Early inadvertent activation of platelets should be avoided because many growth factors have a short half-life and a higher effect can be achieved when activated at or just prior to use (eg, injection).

Some efforts have been made in the preparation or separation of PRP using centrifugation techniques. Examples include the U.S. Patents No. 6,596,180 . 6,610,002 . 6,827,863 . 6,899,813 , US Patent Application Publication No. 2009/0294383 and International Patent Application Publication No. Hei. WO 02/081007 , Unfortunately, known methods have at least one of several disadvantages, including high platelet activation, the inability to remove all or substantially all PRP without risking the aliquoting of a buffy coat or gel with the PRP, or the inability to homogeneity of the PRP and the requirement of sterile open-tube processing.

All publications identified herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated as being incorporated by reference. If a definition or use of a term in a referenced reference is inconsistent with or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference is not applicable.

"Platelet Separation From Whole Blood in an Aqueous Two-Phase System With Water-Soluble Polymers" by Sumida et al., Published in J Pharmacol Sci 101, 91-97 (2006) , recognized a problem of high platelet activation using conventional centrifugation methods and developed a method of separating platelets without centrifuging. More specifically, various polymers were mixed with whole blood containing citrate dextrose and whole blood separation and platelet activation were observed. Unfortunately, Sumida has found that more platelets were activated as the polymers separated the platelets more efficiently. Conversely, there was poor platelet yield when reduced platelet activation was achieved. In addition, due to sedimentation protocols, the platelets are typically not homogeneously distributed in the PRP fractions, and quantification is therefore difficult.

Thus, there is still a need for improved methods of PRP production.

In addition, current separation efforts apparently require aliquoting a PRP (or other fraction of a fluid) from the collection tube in which it is manufactured or separated to transfer to a separate storage container for commercial use. The step of aliquoting may have one or more undesirable effects, including, for example, not aliquoting all of the PRP or other desired fraction, aliquoting unwanted fractions or materials, or contaminating.

Therefore, there is still a need for a method or apparatus that allows the use of a desired fraction of a fluid without the desired fraction from a collection tube it is to be obtained or separated, to be aliquoted for placement in a separate storage container.

Brief description of the invention

The subject invention provides devices in which a fraction of platelet-rich plasma (PRP) having a desired concentration of functioning platelets is separated from a whole blood sample. The separation is achieved by using a polymerizable separation substance which is flowable and curable with whole blood to form a solid barrier.

As used herein throughout the specification and throughout the following claims, the meaning of "a," "an," and "the" includes plural references unless the context clearly dictates otherwise. Also, the meaning of "in" as used herein in the specification includes "in" and "on" unless the context clearly dictates otherwise.

As used herein, a PRP fraction comprises at least 150% of the platelet concentration in the whole blood sample, less than 20% of the white blood cells from the whole blood sample from which it is obtained, and less than 20% of the red blood cells from the whole blood sample from which they are obtained becomes.

The polymerizable separation substance may be contained in a collection tube containing the whole blood sample which is centrifuged until the polymerizable separation substance settles between the PRP fraction and a non-PRP fraction of the whole blood sample. Because the polymerizable release substance (prior to curing) comprises smaller building blocks (eg, oligomers), less friction and shear forces occur between the platelets and the release substance materials, resulting in less platelet activation.

Once the polymerizable separating substance settles between the PRP and non-PRP fractions, the polymerizable separating substance can be hardened to form a solid barrier, the polymerizable separating substance of the solid barrier having an average molecular weight higher than the average molecular weight the polymerizable separation substance is when flowable, and wherein the polymerization does not have a significant adverse effect on the viability of the platelets. The solid barrier is preferably stationary with respect to the collection tube and impermeable to a degree that prevents mixing of the PRP fraction and the non-PRP fraction upon shaking. This advantageously allows a user to homogenize and completely remove the PRP fraction from the collection tube without remixing the PRP and non-PRP fractions.

The subject invention also provides devices in which a PRP fraction or other fraction of another fluid can be used without aliquoting the desired fraction from the collection tube in which it is obtained or separated for placement in a separate storage container have to.

For example, consider an adapter having a needle fluidly coupled to a fluid dispenser (eg, an eye drop dispenser). The needle is sized and dimensioned to pierce at least a portion of the collection tube cap and to contact the desired fraction. The fluid dispenser is located outside of the collection tube when the needle is at least partially disposed in the tube and includes a mechanism that allows or is coupled to the sterile, controlled, dropwise delivery of the fluid fraction.

Various objects, features, aspects and advantages of the subject invention will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawing figures in which like reference numerals represent like components.

Brief description of the drawing

1 FIG. 10 illustrates a collection tube used in accordance with a method of separating a platelet-rich plasma fraction from whole blood. FIG.

2 Figure 3 is a schematic representation of a method for separating a platelet rich plasma fraction from a whole blood sample.

3 shows the "Cts" (Cycle Threshold) of the control, the tube with LAI and the tube with LAIT.

4 shows the non-reproducibility of cfDNA numbers taken from unmixed plasma.

5 shows the aggregation of one control, from LAI and LAIE to ristocetin.

6 shows the aggregation of a control, from LAI and LAIE to collagen.

7 Figure 3 is a schematic representation of another method for separating a platelet-rich plasma fraction from a whole blood sample.

8th Figure 3 is a schematic representation of yet another method for separating a platelet-rich plasma fraction from a whole blood sample.

9A Figure 1 illustrates an embodiment of an adapter of the subject invention.

9B shows an embodiment of a collection tube, which with the adapter 5A can be coupled.

9C is a plan view of a collection tube cap.

Detailed description

The following description provides many exemplary embodiments of the subject invention. Although each embodiment represents a single combination of elements of the invention, the subject invention is intended to encompass all possible combinations of the elements disclosed. Thus, if one embodiment comprises elements A, B and C and a second embodiment comprises elements B and D, then the subject invention is intended to include other remaining combinations of A, B, C or D, although not explicitly disclosed ,

It should be noted that the disclosed techniques provide many advantageous technical effects, including increased platelet separation in the presence of a PSS without substantial activation of the discrete platelets. In addition, the compositions provided enable a user to turn a collection tube upon polymerization of the separation substance to mix the separated PRP fraction without remixing the separated phases or the PSS until a substantially homogeneous distribution of the platelets is achieved. In addition, the devices provided allow the commercial use of a PRP or other fraction of a fluid without removing the fraction from the collection tube in which it was prepared or separated for placement in a separate storage container.

An embodiment for separating a PRP fraction in accordance with the subject invention is schematically illustrated in FIG 1 shown. It is envisaged that any suitable collection tube could be used. The tube is preferably made of a solid material (e.g., hard plastic, glass, etc.) capable of supporting a vacuum in its lumen and having sufficient volume to hold a desirable whole blood sample, from which a desirable PRP Amount can be separated. An exemplary tube contains the products of BD Vacutainer ^® . Although the preferred apparatus and methods described herein involve the use of a collection tube, it is contemplated that a collection tube could be replaced with other containers such as, but not limited to, flasks, jars, cups, bottles or ampoules.

In the embodiment of 1 is a collection tube 100 , which is a flowable / polymerisable separating substance 105 contains, a sterilization energy source 110 exposed to the sterilization energy 115 (eg gamma radiation, e-irradiation, heat) on the tube 100 and the separating substance 105 resulting in a sterilized separation tube and a sterilized (but still flowable and UV-curable) release substance 120 leads. The tube 100 can give a whole blood sample 125 be added, resulting in a mixture 130 from the sample and the sterilized separating substance forms.

The tube 100 and the mixture 130 can then be centrifuged until the separating substance component 140 of the mixture 130 between a PRP group 145 and a non-PRP group 135 the whole blood sample 125 puts. The PRP Group 145 preferably has a platelet concentration that is at least 150% of the platelet concentration of the sample 125 and the non-PRP fraction 135 preferably comprises at least 90% of the WBCs and RBCs from the sample 125 ,

The polymerizable separating substance 140 can be completely or partially polymerized. Therefore, the exposure of the separating substance initiates 140 towards energy 150 (eg, UV energy) generated by a suitable energy source 155 is generated (eg UV light source), a radical polymerization and causes at least part of the separating substance 140 a solid, crosslinked composition 160 forms as an impermeable barrier between the PRP fraction 145 and the non-PRP group 135 acts. In some embodiments, the final thickness of the barrier (after UV curing) is not more than 10 mm, and more preferably not more than 5 mm.

The polymerizable separating substance (or the polymerizable part of a polymerizable separating substance) could be formulated to polymerize within ten minutes to a hardness of at least 1 on the Shore 00 hardness scale, more preferably a hardness of at least 10 on the Shore A hardness scale, when triggered by a suitable source of energy (eg UV light), and a solid relative to a probe, to form a pipette, decanting or even freezing. The formed hardened solid barrier may adhere to the walls of a lumen of a tube to substantially or completely seal the PRP fraction from one or more other fractions, thereby protecting the PRP fraction from contamination by diffusion, agitation, sample extraction, or other undesired interaction becomes.

If a separating substance 140 is only partially polymerizable, it is contemplated that the non-polymerizable moiety (eg, a thixotropic gel component) can settle below the polymerizable moiety such that the desired PRP fraction can be used or removed completely from the collection tube without the To mix PRP fraction with the non-PRP fraction or the non-polymerizable part. The non-polymerizable part could for example Standardgele (z. B. BD Vacutainer ^® SST ^TM, BD Vacutainer ^{® TM} PST, Vacuette ^® blood collection tube with gel racers, PPMA Serumtrenngelröhrchen, Polypropylenserumtrenngelröhrchen etc.) include or other commercially suitable gel.

It should be noted that because of the solid barrier formed, a release substance of the subject invention may allow a user to achieve and re-establish substantial homogeneity of the PRP fraction by mixing or otherwise shaking the PRP fraction in the collection tube, without remove the barrier between the PRP and non-PRP fractions. In addition, substantial homogeneity could be achieved without disrupting the cells, for example, without significant activation of the platelets. In addition, when at least a portion of the separation substance is polymerizable to form a solid, curing of the polymerizable separation substance can form a polymeric network that maintains separation of the PRP fraction as efficiently as, or even better than, a polymeric separation substance.

Some looked polymerizable separating substances may include: (1) an oligomer (e.g., a combination (for example, Ebecryl, Cytec) thereof..) (With (2) a photo initiator, for example, Additol ^® BDK, Additol ^® TPO. ) and (3) a stabilizer (phenothiazine). Examples of photohardenable compositions contemplated include LAI (e.g., L1A1 and phenothiazine) and LAIR. As used herein, L = an oligomer (e.g., L1 = Ebecryl 230 from Allnex, previously from Cytec Industries, Inc., etc.); A = a photoinitiator (eg A1 = Additol BDK); I = a stabilizer (eg phenothiazine etc.). More examples of components that may be included in a PSS are given in Table 1 below. ABBREVIATION SURNAME R Gelling agent (eg DBS, silica gel, etc.) M monomer M1 Trimethylolpropane propoxylate triacrylate L oligomer L1 Ebecryl 230 from Allnex A photoinitiator A1 Additol BDK I stabilizer I1 phenothiazine e Antioxidant / radical scavengers (eg, vitamin E, BHT, BHA, carotene, bilirubin, ascorbic acid, etc.) D Density adjuster (eg Ebecryl 113 from Cytec etc.) T Temponitroxid Table 1

In some polymerizable separation substances contemplated, the photoinitiator is present at a level of less than 10%, less than 5% or even less than 2% by weight, and is a stabilizer at a concentration of less than 5%. %, less than 2% by weight, less than 1% by weight or even less than 0.5% by weight. As a more specific, non-limiting example, a separating substance may be an oligomer present in a concentration between 95 and 100% (100% by weight) of a photoinitiator which is present at a concentration between 0.8 and 1.2% (e.g. 1% by weight) and a stabilizer present in a concentration between 0.01 and 0.05% (eg 0.1% by weight).

Photoinitiators considered include Additol BDK and Additol TPO. Considered stabilizers include phenothiazine, among others. A separation substance may also include an antioxidant, particularly if sterilization of the separation substance collection tube is desired without substantial curing of the separation substance. Considered antioxidants include tocopherol, butylated hydrocytoluene (BHT), butylated hydroxyanisole (BHA), carotene, bilirubin, and ascorbic acid.

An exemplary polymerizable release substance comprises an oligomer, a photoinitiator and a stabilizer wherein the photoinitiator is present at a level of less than 5% and wherein the stabilizer is present at a level of less than 0.5% by weight.

LAI (z. B. L1, A1 and phenothiazine) forms some particularly preferred photocurable compositions and can be formulated so that it has the desired density range of 1.00-1.09 g / cm ^3. Although experiments have been carried out using a photocurable composition with an oligomer, it is contemplated that monomer-containing compositions may also be suitable for use in PRP production because many monomers and oligomers have similar high reactivity and many monomers and oligomers are used in the art Can polymerize the presence of light.

Other examples of potentially suitable photocurable release substances include those described in U.S. Pat U.S. Pat. Nos. 7,674,388 . 7,673,758 . 7,775,962 . 7.971730 . 7,780,861 . 8,206,638 . 8,282,540 . 8,151,996 and 8.318077 LAIE (eg, L1, A1, phenothiazine and tocopherol) and LAIER, where R = a gelling agent (e.g., DBS, silica gel, etc.) and E = at least one of an antioxidant and a scavenger (e.g. Vitamin E, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), carotene, bilirubin, ascorbic acid, etc.). While R and E are generally not required components of a photohardenable composition, each may provide advantageous features for the photohardenable compositions for some uses. For example, a gelling agent is not a required component of the photohardenable composition if the release substance also comprises a thixotropic softening component filled into the collection tube over the photocurable composition such that no flow occurs prior to use. Nevertheless, it may be desirable to have a thixotropic photohardenable composition, for example, if it is desirable to have the sealant in the tube without a softgel component or on the softgel component.

A radical scavenger, such as compounds having vitamin E activity (eg, tocopherol), may not be included to allow the photocurable composition to be sterilized via irradiation without curing (eg, by altering the density properties instead of requiring heat sterilization to maintain flowability effective to allow sedimentation between two fractions of a whole blood sample. If heat sterilization is desired, it can be achieved by exposing the collection tube and separator to a heat of at least 200 degrees Celsius, more preferably at least 225 degrees Celsius, and most preferably at least 250 degrees Celsius.

It is contemplated that an anticoagulant may be included in the collection tube, optionally as part of the separation substance, to prevent coagulation of the whole blood sample and to obtain plasma containing fibrinogen and coagulation factors. Considered anticoagulants include sodium citrate, EDTA, citrate dextrose, or any other anticoagulant. It should be noted that although the Irradiation polymerization is initiated and carried out, the chemistry used, and especially acrylic polymerization, neither platelet activation or function, nor do many or most other tests interfere with the PRP fraction.

2 represents a procedure 200 For separating a PRP fraction from a whole blood sample in a collecting tube with a polymerisable separating substance (PSS). While any suitable amount of the separating substance may be contained in the lumen of the tube, it is preferred that not more than about 1 ml or 2 grams of the separating substance contained per 10 ml volume of the tube.

method 200 involves the step of centrifuging the whole blood sample in the collection tube with the PSS using a centrifugation protocol as in step 210 shown. Consider any suitable centrifugation protocol sufficient to cause the PSS to flow between two fractions of a sample, including, for example, centrifugation for ten minutes at 24 ° C and 1000 rpm as in step 212 shown. Other examples of suitable centrifugation protocols could include: a centrifugation time between one minute and thirty minutes inclusive, at between 500 rpm and 5000 rpm inclusive; or a centrifugation time of between five minutes and twenty minutes at between 700 rpm and 3600 rpm inclusive. As is well known in the art, the centrifugation conditions determine the sedimentation and sedimentation rate of the platelets. One skilled in the art will, without undue experimentation, be able to determine the appropriate applied G forces and the time to achieve a desired yield of platelets above the PSS. Therefore, increasing or reducing the desired yields can be easily achieved using the above considerations.

Unless the context dictates otherwise, all ranges defined herein should be interpreted as including their endpoints, and open ended margins should be interpreted as including only commercially practical values. Likewise, all lists of values should be considered as inclusive, unless the context dictates otherwise.

In most preferred embodiments, the PSS is flowable with the whole blood and has a density between an average density of a PRP fraction and an average density of a non-PRP fraction in accordance with step 215 on.

In order to achieve a desired initial density, it is contemplated that the density of the separating substance may be adjusted by molecular composition or by inclusion of suitable fillers (eg, silica gel, latex, or other inert material).

Additionally or alternatively, centrifuging under a suitable protocol may cause the PSS to be immobilized without substantial activation of platelets in accordance with step 218 between the PRP Group and the non-PRP Group. It is contemplated that when the PSS is used to separate a sample of another fluid or separate a whole blood sample into fractions other than a PRP and non-PRP fraction, it is contemplated that the PSS will have a density between an average density of a first fraction to be separated and an average density of a second fraction to be separated.

As used herein, centrifuging using a first centrifugation protocol to cause the PSS to flow between two fractions of a sample without substantial activation of platelets means that the platelets retain a function within 15%, more preferably within 10% of that of Platelets would be retained in an otherwise identical centrifugation protocol as the first centrifugation protocol in which a PSS is not used (eg, without a separation substance). The essential activation of platelets is measurable, inter alia, based on the ability of platelets in the PRP fraction, non-PRP fraction, or both, to aggregate to at least one of ristocetin and collagen.

The procedure 200 also includes the step 220 curing the PSS without substantial activation of the platelets to form a solid barrier between the PRP fraction and the non-PRP fraction. Depending on the formulation of the separation substance, it is contemplated that the mode or mechanism of polymerization may vary considerably. While discussion herein primarily focuses on UV energy polymerization, all known polymerization processes are considered suitable for use herein. For example, contemplated polymerizations include various radical or cationic polymerizations (eg, using photolabile compounds, radical initiators, etc.), condensation polymerization, esterification, amide formation, and so on. Reactive groups can be acid groups, on most preferably mono- and dicarboxylic groups), conjugated diene groups, aromatic vinyl groups and alkyl (meth) acrylate. The polymerization may advantageously be fully supported by reactive groups of the monomers or oligomers of the separation substance, but additional reagents may also be suitable, including radical initiators.

The step of curing may advantageously form a solid barrier between the PRP fraction and the non-PRP fraction which is stationary with respect to the collection tube. In addition, the solid barrier may be impermeable to a degree that permits agitation of the PRP fraction and the non-PRP fraction as in step 222 shown prevented. The PSS of the solid barrier will have an average molecular weight that is greater than the average molecular weight of the PSS when compared to the whole blood in accordance with step 225 flowable.

As used herein, curing of the polymerizable release substance without substantial activation of platelets means that the platelets have the function within 15%, more preferably within 10%, of what would be retained by platelets when PSS is not used and if there is no step of hardening, retained.

The solid barrier advantageously allows for a step of removing at least 90%, more preferably at least 95% and most preferably 100% of the PRP fraction from the collection tube without remixing the PRP fraction with the non-PRP fraction. This could be accomplished by removing the PRP fraction as it is from the collection tube (eg, by pouring, pipetting, etc.) or by adding saline or other fluid to the tube to remove the entire PRP Support fraction.

method 200 optionally could include a step of homogenizing the PRP fraction, after the curing step, such that various aliquots taken therefrom have platelet counts that are within a coefficient of variation of an applied cell counting method. This can be extremely useful in providing reproducibility and uniformity. Homogenization of the PRP fraction can be achieved using any suitable method, including, for example, mechanical stirring, bubble stirring, inverting the tube, or otherwise agitating or pulverizing through a wide mouth pipette.

platelet recovery

Many in the cosmetics and healthcare industries have found that PRP are optimal with 200-250% platelet concentration of a whole blood sample. Previously used collection tubes (eg, BD Vacutainer with purple cap without separating substance) for obtaining whole blood fractions include centrifuging blood resulting in a serum fraction supernatant, an intermediate buffy layer, and a fraction of red blood cells under the buffy layer. Such tubes and methods make it very difficult or even impossible to remove the supernatant without removing white cells from the buffy coat. On the other hand, when a gel is used, the result is a PRP or serum fraction that depends on the centrifugation protocol, then an intermediate gel layer, then a buffy coat and a bottom RBC fraction. Again, it is very difficult, if not impossible, to remove all of the PRP or serum fraction without aspirating the gel with the PRP or serum. And in any case, there is a significant maldistribution of platelets in the PRP fraction.

The PSS of the subject of the invention advantageously allow platelet recovery at various concentrations (including the range of 200-250%) and also allow the use of all of the PRP supernatant. Table 2 below shows that the platelet yield using LAI or LAIR was at least as good as when using a control. Table 2 also shows that the same red blood cell depletion could be achieved using LAI or LAIR as when using the control. Blue cap (sodium citrate), glass tube, fresh blood, 30 seconds UV exposure Gel Composition = L (100%); A (1.00%); I (0.10%); R (5%) RCF (g) Time (min) Platelets (pre) Platelets (post) % Increase platelets RBC (pre) RBC (Post) % RBC depletion Control 1 300 5 198 435 220 4.19 0.03 99 Control 2 300 5 216 462 214 4.71 0.04 99 LAI 1 300 5 205 381 186 4.22 0.03 99 LAI 2 300 5 194 392 202 4.16 0.03 99 LAIR 1 300 5 192 480 250 4.18 0.03 99 LAIR 2 300 5 201 469 233 4.17 0.06 99 Red cap, glass tube, concentrated blood, 30 seconds UV exposure Gel Composition = L (100%); A (1.00%); I (0.10%); R (5%) RCF (g) Time (min) Platelets (pre) Platelets (post) % Increase platelets RBC (pre) RBC (Post) % RBC depletion Control 1 300 5 193 333 173 1.96 0.03 98 Control 2 300 5 122 186 152 2.05 0.04 98 LAI 1 300 5 117 175 150 2.02 0.03 99 LAI 2 300 5 141 267 189 2.73 0.03 99 LAIR 1 300 5 114 197 173 2.11 0.03 99 LAIR 2 300 5 121 195 161 2.13 0.06 97 Blue cap (sodium citrate), glass tube, concentrated blood, 30 seconds UV exposure Gel Composition = L (100%); A (1.00%); I (0.10%); R (5%) RCF (g) Time (min) Platelets (pra) Platelets (post) % Increase platelets RBC (pre) RBC (Post) % RBC depletion Control 1 200 12 121 155 128 2.20 0.03 99 Control 2 200 12 143 165 115 2.08 0.03 99 LAI 1 200 12 134 164 122 2.19 0.03 99 LAI 2 200 12 138 164 119 2.14 0.03 99 LAIR 1 200 12 122 161 132 2.18 0.03 99 LAIR 2 200 12 122 169 139 2.07 0.03 99 Blue cap (sodium citrate), glass tube, concentrated blood, 30 seconds UV exposure Gel Composition = L (100%); A (1.00%); I (0.10%); R (5%) RCF (g) Time (min) Platelets (pre) Platelets (post) % Increase platelets RBC (pre) RBC (Post) % RBC depletion LAI 1 150 10 171 247 144 2.58 0.04 98 LAI 2 150 10 177 211 119 2.04 0.05 98 LAIR 1 150 10 187 228 122 1.8 0.05 97 LAIR 2 150 10 198 248 125 1.54 0.05 97 Blue cap (sodium citrate), glass tube, concentrated blood, 30 seconds UV exposure Gel Composition = L (100%); A (1.00%); I (0.10%); R (5%) RCF (g) Time (min) Platelets (pre) Platelets (post) % Increase platelets RBC (pre) RBC (Post) % RBC depletion LAI 1 150 20 183 243 133 2.39 0.03 99 LAI 2 150 20 142 139 98 1.69 0.02 99 LAIR 1 150 20 123 150 122 1.62 0.02 99 LAIR 2 150 20 128 146 114 1.6 0.02 99 Table 2

Table 3 below shows that the platelet yield using LAI or LAIE was at least as good as when using a control. Table 2 also shows that the same or a Similar depletion of white blood cells using LAI or LAIE could be achieved as using the control.

The tested compositions (LAI and LAIE) consist of the following: LAI LAYMAN L 100% 100% A 1% 1% I 0.10% 0.10% e - 200 mM

The manual differential of LAI was as follows: 8% neutrophils, 80% lymphocytes, 10% monocytes, 2% eosinophils; The manual differential of LAIE was as follows: 5% neutrophils, 89% lymphocytes, 5% monocytes, 1% eosinophils. Sample ID Control 1 WB Control 2 WB Avg. control Control 1 plasma Control 2 plasma Avg. Control plasma Control 1 UV Exp Centrifuge No No Yes Yes Yes UV exposure No No No No 1:45 min at 1 '', 25% WBC [10 ^ 3 / uL] 3.6 3.66 3.63 0 0.01 0.005 0.10 RBC [10 ^ 6 / uL] 3.97 3.93 3.95 0.02 0.02 0.02 0.01 HGB [g / dL] 12.8 12.6 0 0 0.00 HCT [%] 38.1 37.6 0.1 0.1 0.10 MCV [fL] 96 95.7 50 50 100.00 MCH [pg] 32.2 32.1 0.0 0.0 0.0 MCHC [g / dL] 33.6 33.5 0.0 0.0 0.0 RDW-SD [fL] 45.7 45.8 - - - RDW-CV [%] 12.8 13 - - - PLT [10 ^ 3 / uL] 130 139 134.5 182 156 186.00 MPV [fL] 11.3 11 11 10.8 10.50 Control 2 Exp UV Avg. Control Exp UV LAI plasma % Depletion LAIE plasma Yes Yes Yes 1:45 min at 1 '', 25% 01:00 min at 1 '', 25% 01:45 min at 1 '', 25% 0.16 0.13 0.06 98.3 0.06 98.3 0.01 0.01 0.03 99.2 0.02 99.5 0 0 0 0.1 0.1 0.1 100 33.3 50 0.0 0.0 0.0 0.0 0.0 0.0 - - - - Platelet factor increase 177 301 2.2 275 2.0 10.5 11.2 11 Table 3

Table 4 shows the platelet yield and depletion of white blood cells under a centrifugation protocol as follows: 1000 rpm for 10 minutes at 24 ° C. There was a slight increase in platelet yield and a similar depletion of white blood cells / red blood cells.

The tested compositions (LAI and LAIE) consist of the following: LAI LAYMAN L 100% 100% A 1% 1% I 0.10% 0.10% e - 200 mM

The experiments were carried out in sodium citrate tubes. Sample ID Control 1 Control 2 Avg. controls LAI 1 Control 1 plasma Control 2 plasma Centrifuge No No No Yes Yes UV exposure No No No No No WBC [10 ^ 3 / uL] 6.86 6.46 6.66 6.45 0.09 0.03 RBC [10 ^ 6 / uL] 4.17 4.22 4.20 4.35 0.01 0.01 HGB [g / dL] 12.9 13 13.0 13.2 0 0 HCT [%] 38.3 38.9 38.6 40.3 0.1 0.1 MCV [fL] 91.8 92.2 92.0 92.6 100 100 MCH [pg] 30.9 30.8 30.9 30.3 0.0 0.0 MCHC [g / dL] 33.7 33.4 33.6 32.8 0.0 0.0 RDW-SD [fL] 44.2 44 44.1 44.9 - - RDW-CV [%] 13.2 13.2 13.2 13.2 - - PLT [10 ^ 3 / uL] 226 215 220.5 226 385 400 MPV [fL] 8.9 8.8 8.9 8.4 9.4 9.3 Avg. Control plasma Post-UV Control 1 Post-UV Control 2 Avg. Post-UV control LAI 1 plasma Yes Yes Yes 1:45 min at 1 '', 25% 01:45 min at 1 '', 25% 01:00 min at 1 '', 25% 0.06 0.10 0.07 0.09 0.21 0.01 0.02 0.02 0.02 0.02 0 0.00 0 0 0 0.1 0.10 0.1 0.1 0.1 100 50,00 50 50 50 0 0.0 0.0 0 0.0 0 0.0 0.0 0 0.0 - - - - - - 392.5 336.00 393 364.5 449 9.35 9.90 9.5 9.7 8.9 Sample ID LAI 2 plasma Avg. LAI LAIE 1 plasma LAIE 2 plasma Avg. LAYMAN Ctrl Depletion RBC, WBC % Depletion LAI % Depletion LAIE Centrifuge Yes Yes Yes UV exposure 01:00 min at 1 '', 25% 01:00 min at 1 '', 25% 1:45 min at 1 '', 25 WBC 0.15 0.18 0.23 0.02 0,125 99,10 97.30 98.12 RBC 0.01 0,015 0.02 0.01 0,015 99.76 99.64 99.64 HGB [g / dL] 0 0 0 0 0 HCT [%] 0 0.05 0.1 0 0.05 MCV [fL] 0 25 50 100 75 MCH [pg] 0.0 0 0.0 0.0 0.0 MCHC [g / dL] - 0.0 0.0 0.0 RDW-SD [fL] - - - RDW-CV [%] - - - Factor platelets increased PLT [10 ^ 3 / uL] 394 421.5 456 439 447.5 1.8 1.9 2.0 MPV [fL] 8.8 8.85 9 9 9 Table 4

Obtaining a homogeneous PRP fraction

The PSS of the subject invention provides an additional advantage of enabling production of a substantially homogeneous PRP (or other) fraction. Applicant has compared the recovery of cell-free DNA in 3 tube types and found that aliquots taken from a non-mixed sample would give variable results. From a different perspective, aliquots will be highly variable if a user aliquots plasma from a non-gel tube without mixing. If a user aliquots the plasma from the tube and mixes it outside the tube, the results may be more reproducible, but the recovery is worse.

The 3 tube types tested are as follows: Control ("no gel"), tubes with LAI Photogel and tubes with LAIT photogel, where L = oligomer, A = photoinitiator Additol BDK, I = stabilizer phenothiazine and T = temponitroxide. 3 shows the "Cts" (Cycle Threshold) of the control, the tube with LAI and the tube with LAIT. The lower Ct of LAI and LAIT compared to the control indicates a better recovery compared to the control. 5 also shows that the results using LAI and LAIT were as reproducible as the control. N = 4 for each tube type.

The protocol used is as follows: Concentrated blood was mixed and added with a pipette to each of the nine tubes. The photogel tubes were pre-filled with either LAI or LAIT. All tubes were centrifuged at room temperature for 10 minutes at 3000 rpm. After centrifugation, the photogel tubes were exposed to UV light in a light box for 1 minute. The plasma from each tube was removed and for tubes with the solidified barrier all plasma was removed. Care was taken not to destroy the cell layer for the control gel without gel, which required some plasma to be left behind (approximately 100 uL). The plasma was sent to the DNA molecular biology lab for molecular pathology. There, the four tubes were further aliquoted into three samples each. Thus, the variability shown involves the process of dividing the plasma from each tube in three ways

Plasma obtained from an EDTA tube without gel was tried manually on the upper layer, lower (near the cell) layer and the middle section. The plasma was mixed by vortexing and tried again ("mixed"). The melting curves shown below demonstrate that the recovered cfDNA varies with manual sampling. Cell-free DNA was used as a proximal marker for platelet production.

In the in 4 As shown, the aliquot taken from the bottom layer of the unmixed plasma had more cfDNA than the aliquot taken from a central section or blended upper section (deeper well indicates greater recovery).

Since the methods described herein involve the use of a PSS that is hardened to form a solid barrier, the PRP fraction may be mixed in the collection tube in which it is obtained or separated, thus homogenizing the PRP fraction in the collection tube Collection tube allows without being exposed to an environment outside the collection tube. It should be noted that the PSS allows a user to obtain a homogeneous reliable platelet count since amounts obtained from different portions of the tubule can be within a coefficient of variation of an applied cell count method.

Plättchenviabilität

As discussed above, Applicants were able to show that using PSSs of the subject invention, a similar or even improved platelet recovery could be achieved. It is known in the art that although solid barriers with long polymers often improve platelet recovery, they interfere with platelet function. The experiments and data provided below demonstrate that applicant's PSSs allow for optimal platelet harvesting while maintaining viability of the platelets.

Platelet viability was tested by observing platelet aggregation to specific agonists on the Chrono-Log Aggregometer. The PRP was filled by centrifuging whole blood, which was added to a control tube (BD sodium citrate tube, no gel), LAI tube (BD sodium citrate to which LAI was added) and LAIE tube (BD sodium citrate tube to which LAIE was added) , receive. The photogel tubes were exposed to UV light after centrifuging to solidify the barrier. The PRP was aliquoted and diluted with platelet-poor plasma to a platelet concentration required for the standard chrono-log procedure. The following are exemplary curves of platelet aggregation to ristocetin and collagen. Collagen is a potent agonist that induces aggregation, secretion of platelet granules and thromboxane synthesis, thus it is an inclusive test for platelet viability.

As in 5 shown, there was no significant aggregation difference between the control, LAI and LAIE.

As in 6 No significant difference is seen in platelet-to-collagen platelet aggregation curves in control, LAI or LAIE curves, indicating that LAI and LAIE do not significantly affect platelet functionality.

7 represents another procedure 700 for separating a PRP fraction from a whole blood sample in a collection tube with a PSS that is flowable with whole blood and has a density between an average density of a PRP fraction and an average density of a non-PRP fraction of whole blood.

method 700 involves the step of centrifuging the whole blood sample in the collection tube with the PSS using a centrifugation protocol as in step 710 shown. Similar to the step of centrifuging from procedures 200 If centrifugation, using a suitable centrifugation protocol, will cause the PSS to undergo no substantial activation of platelets as in step 712 shown flowing between the PRP fraction and the non-PRP fraction. An essential activation is measurably based on the ability of the platelets in the PRP fraction to at least one of ristocetin and collagen as in step 715 shown to aggregate.

The procedure 700 further includes the step of curing the PSS without substantial activation of the platelets to form a solid barrier between the PRP fraction and the non-PRP fraction as in step 720 shown. The PSS of the solid barrier will have an average molecular weight that is greater than the average molecular weight of the PSS when compared to the whole blood in accordance with step 725 flowable (eg before the hardening step).

In some preferred methods, the solid barrier, after exposure to UV energy, relative to the collection tube at an intermediate position below the PRP fraction and above the non-PRP fraction is stationary and impermeable to a degree that permits mixing of the PRP Group and non-PRP faction in agitation as in step 722 shown prevented. The solid barrier allows removal of at least 90%, more preferably at least 95%, and most preferably 100% of the PRP fraction from the collection tube. From a different perspective, the process involves 700 an optional step of removing at least 95% of the PRP fraction from the collection tube in accordance with step 730 ,

The PRP fraction may advantageously comprise not more than 10% of white blood cells from the whole blood sample. Additionally or alternatively, it is contemplated that no more than 25% of the white blood cells in the PRP fraction will be granulocytes.

The procedure 700 could also include homogenizing the PRP fraction in the collection tube after the hardening step. The homogenizing step could be carried out immediately after curing, within one hour after curing, within one day after curing, at least two days after curing, at least five days after curing, or even at least one month after curing. Homogenizing the PRP fraction results in a substantially homogeneous PRP fraction such that various aliquots taken therefrom have platelet counts that are within a coefficient of variation of an applied cell counting procedure.

8th represents another method 800 for separating a PRP fraction from a whole blood sample in a collection tube with a PSS that is flowable with whole blood and has a density between an average density of a PRP fraction and an average density of a non-PRP fraction of whole blood.

It should be noted that a PRP fraction could have any suitable platelet concentration that is greater than a platelet concentration of a whole blood sample from which it is obtained or separated. For example, a PRP fraction could comprise a platelet concentration of at least 150%, at least 200%, at least 250%, or even higher, a platelet concentration of the sample from which it is obtained. From another perspective, a PRP fraction could include a platelet concentration between 180% and 260% or between 200% and 250% of a platelet concentration of the sample from which it is obtained.

The procedure 800 includes a step of centrifuging a whole blood sample in a collection tube with a PSS and under a centrifugation protocol as in step 810 shown. Centrifugation under the first protocol may cause the PSS to flow between the PRP fraction and the non-PRP fraction, preferably without substantial activation of platelets as in step 812 shown.

The procedure 800 also includes a step of exposing the collection tube, after centrifuging, to UV energy for a period of less than ten minutes to polymerize the PSS and form a solid barrier as in step 820 shown. The PSS of the solid barrier will have an average molecular weight greater than the average molecular weight of the PSS when compared to the whole blood sample as in step 822 shown flowable.

The procedure 800 further includes a step of homogenizing the PRP fraction in the presence of the solid barrier to form a substantially homogeneous PRP fraction so that different aliquots taken therefrom have platelet counts that are within a coefficient of variation of an applied cell counting method.

The methods described herein advantageously enable a user to separate fractions having different densities of a sample (e.g., blood) contained in a collection tube with a PSS. The solid barrier allows for both homogeneity and reproducibility and Applicant has found that by providing an adapter so that the tubes in which the fractions are separated need never be opened, further advantages could be achieved.

9A - 9C represents a collection device 900 and an adapter 950 which can be used with the collection device to allow closed processing while maintaining sterility through multiple uses of a fluid contained therein, resulting in a lower risk of contamination. It should be noted that the adapter 950 could be used not only with PRP applications, but also with another fluid (eg, serum separated from a whole blood sample, plasma separated from a whole blood sample, etc.) stored in a collection device (e.g., a collection tube or other suitable container) is processed.

The collection device 900 includes a tube section 930 and a tube cap section 920 who cooperate to get a fluid 910 (here a PRP fraction), and optionally a solidified PSS and non-PRP fraction, from an environment outside the collection tube. The tube cap section 920 includes a hard material 922 (For example, a plastic), a rubber or other material 925 surrounds the one by the needle 960 of the adapter 950 is penetrable.

The needle 960 is sized and dimensioned to include at least a portion of the tube cap portion 920 pierces and the fluid 910 contacted in the tube. The needle 960 is to a fluid dispenser 970 which is configured as an eye dropper and positioned outside of the collection device when the needle is at least partially in the tube. The fluid dispenser 970 includes a mechanism 980 or coupled thereto (eg, bulb, valve, etc.) that allows for sterile, controlled, dropwise delivery of the fluid from the collection device.

As used herein and unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (with two elements coupled together) and indirect coupling (with at least one additional element between the two elements located). Therefore, the terms "coupled to" and "coupled to" are used interchangeably.

A fluid dispenser configured as an eye dropper may include a small tube and a vacuum bulb. The eye dropper can be configured to be fluid 910 from a first end (eg needle 960 ) and the fluid 910 from a second end (eg tip of the fluid dispenser 970 ) dispenses drop by drop or donates. In some embodiments, saline or other fluid (s) may be fluid 910 be injected into the collection device or otherwise placed before the tube cap 920 with the needle 960 is punctured. The PRP, with or without saline solution, could be used to treat or prevent dry eye syndrome or other eye diseases. It should be noted that an eye dropper is a device configured to contain a fluid and deliver the fluid in a controlled, droplet fashion directly to an user's eye.

An alternative example of a contemplated fluid dispenser is a needle that may be coupled to a syringe for injectable applications.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group element may be referenced and claimed individually or in combination with other elements of the group or other elements found therein. One or more elements of a group may be included in or deleted from a group for convenience and / or patentability. When such inclusion or erasure occurs, the specification herein is intended to include the group as modified, thereby satisfying the written description of all of the Markush groups used in the appended claims.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group element may be referenced and claimed individually or in combination with other elements of the group or other elements found therein. One or more elements of a group may be included in or deleted from a group for convenience and / or patentability. When such inclusion or erasure occurs, the specification herein is intended to include the group as modified, thereby satisfying the written description of all of the Markush groups used in the appended claims.

It should be apparent to those skilled in the art that many modifications are possible, other than those already described, without departing from the concepts of the invention herein. The subject of the invention should therefore not be limited except as defined in the appended claims. In addition, in interpreting the specification and claims, all terms should be interpreted in the most comprehensive manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring in a non-limiting manner to elements, components or steps, indicating that the elements, components or steps referred to are associated with other elements, components or steps not expressly referred to, available, used, or combined. If the claims of the patent refer to at least one of something selected from the group consisting of A, B, C ..., and N, the text should be interpreted to require only one member of the group , not A plus N or B plus N, etc.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 6596180 [0004]
• US 6610002 [0004]
• US 6827863 [0004]
• US 6899813 [0004]
• WO 02/081007 [0004]
• US 7674388 [0043]
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Cited non-patent literature

• "Platelet Separation From Whole Blood in An Aqueous Two-Phase System With Water-Soluble Polymers" by Sumida et al., Published in J Pharmacol Sci 101, 91-97 (2006) [0006]

Claims (10)

A sample collection tube for separating a PRP fraction of whole blood, comprising: a tube with a lumen; a polymerizable composition disposed in a lumen of the tube, the polymerizable composition comprising an oligomer, a photoinitiator, and a stabilizer; wherein the polymerizable composition is flowable with the whole blood, has a density between an average density of a PRP fraction and an average density of a non-PRP fraction, and a first average molecular weight; wherein the polymerizable composition is formulated such that it can be centrifuged with the tube to settle between the PRP fraction and a non-PRP fraction without substantial activation of platelets in the PRP fraction, wherein substantial activation based on the Ability of the platelets in the PRP fraction to aggregate to at least one of ristocetin and collagen is measurable. The sample collection tube of claim 1, wherein the polymerizable composition is further formulated to harden when exposed to UV energy to form a solid barrier within ten minutes. The sample collection tube of claim 2, wherein the solid barrier has a second average molecular weight that is greater than the first average molecular weight. The sample collection tube of claim 3, wherein the solid barrier enables removal of at least 95% of the PRP fraction from the collection tube without remixing. The sample collection tube of claim 4, wherein the PRP fraction in the collection tube can be agitated and shaken at least one of without removing the solid barrier. The sample collection tube of claim 1, wherein the polymerizable composition is formulated so that it can be centrifuged without significant activation of platelets in the PRP fraction for between 5 and 20 minutes and between 700 and 3600 rpm. The sample collection tube of claim 1, wherein the polymerizable separation substance comprises a radical scavenger having vitamin E activity and is formulated such that it can be sterilized by irradiation and maintains flowability with the whole blood. The sample collection tube of claim 1, wherein the photoinitiator is present in a concentration of less than 5% and wherein the stabilizer is present in a concentration of less than 0.5% by weight of the polymerizable composition. The sample collection tube of claim 1, wherein the polymerizable separation substance further comprises a gelling agent. The sample collection tube of claim 1, further comprising a thixotropic gel disposed in the lumen of the tube, and wherein the thixotropic gel moves below the polymerizable composition upon centrifugation from above the polymerizable composition.

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