Cosmetic methods and products

Abstract

This invention relates to method of improving the appearance of skin or hair. In particular, it relates to cosmetic products such as creams and serums comprising a subject's own platelet rich plasma (PRP). The invention also relates to kits that allow the user to apply the cosmetic products daily for several days.

Claims

1. A method of improving the cosmetic appearance of skin of a subject, wherein the method comprises: a) providing a sample of platelet rich plasma from the subject, wherein the platelet rich plasma comprises more than 500,000 platelets/l; b) providing an activator; c) admixing a portion of the platelet rich plasma and a portion of the activator to form a topical composition; d) applying the topical composition to an area of the subject's skin less than 4 hours after the platelet rich plasma and the activator have been admixed to form the topical composition; and e) repeating steps (c) and (d) at least once a day for at least two days.

2. The method of claim 1, wherein the topical composition is applied to an area of the subject's skin less than 1 hour after the platelet rich plasma and the activator have been admixed to form the topical composition.

3. The method of claim 2, wherein the topical composition is applied to an area of the subject's skin less than 10 minutes after the platelet rich plasma and the activator have been admixed to form the topical composition.

4. The method of claim 3, wherein the topical composition is applied to an area of the subject's skin less than 2 minutes after the platelet rich plasma and the activator have been admixed to form the topical composition.

5. The method of claim 1, wherein steps (c) and (d) are repeated for at least three days.

6. The method of claim 5, wherein steps (c) and (d) are repeated at least once a day for at least eight days.

7. The method of claim 1, wherein the area of the subject's skin has been treated using PRP injection or another cosmetic treatment.

8. The method of claim 1, wherein the platelet rich plasma comprises more than 900,000 platelets/l.

9. The method of claim 1, wherein each time a portion of the platelet rich plasma is admixed with a portion of the activator to form a topical composition growth factors are released from platelets in the platelet rich plasma so that fresh growth factors are applied to the skin each time the topical composition is applied to the skin.

10. The method of claim 1, wherein the activator does not comprise calcium chloride.

11. The method according to claim 1, wherein the activator is collagen.

12. The method according to claim 1, wherein the platelet rich plasma is stored for at least 1 day before it is admixed with the activator to form a topical composition.

13. The method according to claim 1, wherein the activator is disposed in a cosmetically acceptable carrier.

14. The method according to claim 13, wherein the cosmetically acceptable carrier comprises a cream, gel, serum, balm, sun cream, after sun cream, foundation, tinted cream, tinted sun cream, soothing anti redness cream with green tint, eyelash gel, lip balm, scalp serum, solution, suspension, emulsion, ointment, foam, paste, lotion, powder, soap, surfactant-containing cleansing oil or spray, or a combination thereof.

15. The method according to claim 1, wherein the sample of platelet rich plasma does not have an activator added to it prior to step (c).

Description

(1) There now follows by way of example only a detailed description of the present invention with reference to the accompanying drawings, in which;

(2) FIG. 1 shows a view of an embodiment of a container of the present invention.

(3) FIG. 2 shows a view of an embodiment of a container of the present invention, the whole container is shown in FIG. 2a, embodiments of a chamber suitable for containing PRP is shown in FIGS. 2b-d.

(4) FIG. 3 shows an embodiment of a chamber suitable for containing PRP.

(5) FIG. 4 shows pictures of the treated areas of patients 1, 2 and 3 from group 1 and patients 1, 2 and 3 from group 2 showing a comparison of the skin before the treatment and after the treatment. Group 1 had PRP injections in the treated area followed by using serum mixed with PRP each day for 16 days. Group 2 only used serum mixed with PRP each day for 24 days on one side of their face (fresh blood was taken every 8 days) Group 3-used the collagen serum only (without adding PRP to it).

(6) FIG. 5 shows Light transmittance platelet aggregometry. The aggregometer measures changes in light transmission of a stirred platelet suspension exposed to a platelet agonist. It is calibrated by a cuvette containing platelet poor plasma which equates to 100% light transmission. Platelet rich plasma stirred in the absence of platelet agonist equates to 0% light transmission (baseline, A). Change in platelet shape due to platelet activation induced by addition of a platelet agonist results in a short decrease in the light transmission (B). When platelet aggregates are forming an increase in the light transmission is recorded by the device and calculated as percentage of aggregation (C).

(7) FIG. 6 shows the PRP storage containers: (a) 70 mL Nunc flask (smallest shown) (b) 30 mL Sterilin universal,

(8) FIG. 7 shows Luckham R100 Rotatest Shaker

(9) FIG. 8 shows the ELISA plate is provided already coated with a capture antibody directed against human PDGF-BB. The PDGF-BB present in test samples or standards, the target protein, binds to the capture antibodies and becomes anchored to the plate. A second antibody directed against human PDGF-BB that is conjugated to Biotin is added which binds to the PDGF-BB captured by the first antibody during a two hour incubation. Residual, unbound material is washed off and streptavidin conjugated to the enzyme horse radish peroxidise (HRP) is added to each well. Streptavidin forms strong, non-covalent 1:1 complexes with biotin during a two hour incubation. Residual material is washed off and a substrate for the enzyme, tetramethyl-benzidine, is added. The product of the enzyme-substrate reaction is coloured, the intensity of which is directly proportional to the amount of PDGF-BB in the samples, which is assessed against a standard curve. Diagram reproduced from www.mitosciences.com

(10) FIG. 9 shows Collagen-induced aggregation: Day 1. Trace 1 and Trace 3: 10 g/mL collagen. Trace 2 and Trace 4: 5 g/mL collagen,

(11) FIG. 10 shows Collagen-induced aggregation for PRP stored in Nunc flasks: Day 2, Trace 1 and Trace 2 Constant agitation MA: 27% Plt: 257109/L Trace 3 and Trace 4 Intermittent agitation MA: 52% Plt: 241109/L Trace 5 and Trace 6 Stationary MA: 65% Plt: 228109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 29% Plt: 243109/L

(12) FIG. 11 shows collagen-induced aggregation for PRP stored in universals: Day 2 Trace 1 and Trace 2 Constant agitation MA: 79% Plt: 149109/L Trace 3 and Trace 4 Intermittent agitation MA: 92% Plt: 221109/L Trace 5 and Trace 6 Stationary MA: 101% Plt: 215109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 77% Plt: 149109/L

(13) FIG. 12 shows collagen-induced aggregation for PRP stored in Nunc flasks: Day 3, Trace 1 and Trace 2 Constant agitation MA: 19% Plt: 245109/L Trace 3 and Trace 4 Intermittent agitation MA: 36% Plt: 202109/L Trace 5 and Trace 6 Stationary MA: 40% Plt: 213109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 12% Plt: 221109/L

(14) FIG. 13 shows collagen-induced aggregation for PRP stored in universals: Day 3, Trace 1 and Trace 2 Constant agitation MA: 61% Plt: 128109/L Trace 3 and Trace 4 Intermittent agitation MA: 88% Plt: 221109/L Trace 5 and Trace 6 Stationary MA: 97% Plt: 233109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 47% Plt: 145109/L

(15) FIG. 14 shows collagen-induced aggregation for PRP stored in Nunc flasks: Day 4, Trace 1 and Trace 2 Constant agitation MA: 15% Plt: 223109/L Trace 3 and Trace 4 Intermittent agitation MA: 23% Plt: 213109/L Trace 5 and Trace 6 Stationary MA: 37% Plt: 209109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 15% Plt: 213109/L

(16) FIG. 15 shows collagen-induced aggregation for PRP stored in universals: Day 4, Trace 1 and Trace 2 Constant agitation MA: 43% Plt: 117109/L Trace 3 and Trace 4 Intermittent agitation MA: 65% Plt: 236109/L Trace 5 and Trace 6 Stationary MA: 77% Plt: 243109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 34% Plt: 133109/L

(17) FIG. 16 shows collagen-induced aggregation for PRP stored in Nunc flasks: Day 5, Trace 1 and Trace 2 Constant agitation MA: 13% Plt: 205109/L Trace 3 and Trace 4 Intermittent agitation MA: 18% Plt: 193109/L Trace 5 and Trace 6 Stationary MA: 30% Plt: 183109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 11% Plt: 200109/L

(18) FIG. 17 shows Collagen-induced aggregation for PRP stored in universals: Day 5, Trace 1 and Trace 2 Constant agitation MA: 61% Plt: 102109/L Trace 3 and Trace 4 Intermittent agitation MA: 64% Plt: 222109/L Trace 5 and Trace 6 Stationary MA: 75% Plt: 233109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 27% Plt: 118109/L

(19) FIG. 18 shows Collagen-induced aggregation for PRP stored in Nunc flasks: Day 8 Trace 1 and Trace 2 Constant agitation MA: 6% Plt: 191109/L Trace 3 and Trace 4 Intermittent agitation MA: 12% Plt: 151109/L Trace 5 and Trace 6 Stationary MA: 20% Plt: 146109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 6% Plt: 186109/L

(20) FIG. 19 shows Collagen-induced aggregation for PRP stored in universals: Day 8 Trace 1 and Trace 2 Constant agitation MA: 58% Plt: 85109/L Trace 3 and Trace 4 Intermittent agitation MA: 39% Plt: 188109/L Trace 5 and Trace 6 Stationary MA: 37% Plt: 208109/L Trace 7 and Trace 8 Constant agitation+PGE1 MA: 27% Plt: 87109/L

(21) FIGS. 20 and 21 plot the changes in final percentage collagen-induced aggregation over time in PRP variably stored in Nunc flasks and universal containers respectively

(22) FIGS. 22 and 23 plot the changes in platelet count over time in PRP variably stored in Nunc flasks and universal containers respectively

(23) FIG. 24 shows Standard curve for PDGF-BB ELISA,

(24) FIG. 25 shows Plots of supernatant PDGF-BB levels count over time in variably stored PRP FIGS. 25 and 26 plot the PDGF-BB levels in supernatants of collagen-activated PRP that had been variably stored in Nunc flasks and universal containers respectively. Serial PDGF-BB levels for supernatants of Nunc flask-stored PRP after collagen activation. Note that a nominal level has been entered for the unavailable Day 2 result of constant agitation+PGE1 to permit full graphical plot

(25) FIG. 26 shows Serial PDGF-BB levels for supernatants of universal-stored PRP after collagen activation. Note that a nominal level has been entered for the unavailable Day 2 result of constant agitation to permit full graphical plot.

(26) FIG. 27 shows blood that has been separated by centrifugation. Starting from the bottom of each tube the first layer is Erythrocytes, the second very thin layer is of Leukocytes, above the leukocytes is the F2 fraction of the plasma (also called PRP) (about 50% of the plasma) and right on the top is the F1 fraction of the plasma (also called PPP) (the other 50% of the plasma). It is the F2 (PRP) that was used in the tests on platelet storage conditions that are described in this application and it is also the F2 (PRP) fraction that was used in the clinical tests described in this application. F2 fraction is slightly darker than F1 fraction which makes it possible for a practitioner to draw up the F1 fraction first and then F2 fraction separately. The plasma fraction is marked. The plasma fraction is separated into Fraction 1 (F1) (PPP) and Fraction 2 (F2) (PRP). F2 is richer in platelets than F1 and therefore F2 is preferably used in the methods of the present invention.

(27) FIG. 28 shows blood that has been separated by centrifugation. Fraction 1 of the plasma (F1) (also called PPP) is marked.

(28) FIG. 29 shows blood that has been separated by centrifugation where the F1 fraction has been removed leaving the F2 fraction,

(29) FIG. 30 shows 4 views, from top left clockwise: inside; front; back and front open, of the centrifuge used to separate blood to provide F1 and F2 fractions of plasma. The centrifuge is a BTI-Endornet centrifuge which spins the blood at 580 G for 8 minutes. During the centrifugation process blood gets separated into the fractions described in FIGS. 27 to 29.

(30) FIG. 31 shows a kit according to the present invention. The kit comprises: a container that is sterile inside for keeping the plasma, PRP or F2 in, this is shown at the right hand side of the figure; several ampules containing the cosmetically acceptable composition, there may be one ampule for each application of the composition to the skin, the ampules are shown at the top of the figure; an agitating device which can be used to constantly agitate the plasma, PRP or F2 inside its container, this is shown at the bottom of the picture.

(31) The cosmetic composition may be: Sun cream, After sun, Foundation, Tinted cream, tinted sun cream, Soothing anti redness cream with the green tint to mask redness of the skin, Scalp serum.

(32) The Growth Factors:

(33) For any PRP, 7 fundamental protein growth factors are concentrated in the plasma after centrifuging (to what degree and with what level of undesirable components will vary) Platelet-derived growth factorsPDGF-AA, PDGFAB, PDGFBB Transforming growth factorsTGFB1, TGFB2 Vascular endothelial growth factorsVEGF Epithelial growth factorsEGF

(34) This concentrate also contains 3 proteins in the blood to act as cell adhesion molecules: fibrin, fibronectin, and vitronectin.

(35) PDFG, VEGF and EGF are the most important for new tissue regeneration. The carrier may comprising one or more ingredients selected from table 1.

(36) The carrier may comprise

(37) TABLE-US-00001 TABLE 1 All items stated include both their Organic and Synthetic versions. Item Function D522 (Dry Flo Plus) Absorbant D572 (Dry Flo AF) Absorbant K505 (Kaolin 2747) Absorbant M742 (Magnesium Carbonate) Absorbant S813 (Sorbosil BFG50) Absorbant C936 (Color Clay Rosa) Absorbant/abrasive A577-CC (Alistin P5) Active against cell membrane damage L592 (Latex LATZ) Adhesive polymer G513 (Glypure 70) AHA, pH adjuster L546 (Lactic Acid 90%) AHA, pH adjuster Z503 (Zincidone) Anti dandruff A764 (Avenacare Beta Glucan) Anti oxidant D571-HM (Deepaline PVB) Anti wrinkle active M704-CC (Matrixyl 3000) Anti wrinkle active S759 (Syn-tacks) Anti wrinkle active S761 (Syn-Ake) Anti wrinkle active C911 (Symglucan) Anti-ageing active O581-CC (Oxygen Complex LS 9641) Anti-ageing active C642 (Cabosil M5) Anticaking agent V506 (Veegum Ultra) Anticaking agent P564 (Protaderm HA) Antidandruff active S559 (Sodium Salicylate) Antimicrobial S965 (Sodium Sulfite) Antimicrobial B507 (BHT) Antioxidant O510 (Oxynex K Liquid) Antioxidant P835 (Phyto Terra Organic Mate) Antioxidant S578 (Stay C50) Antioxidant V501 (Vitamin E Acetate (dl-alpha Antioxidant Tocopheryl Acetate)) V528 (Vitamin A Palmitate (1.7 iu)) Antioxidant V529 (Vitamin A Palmitate (1.0 ml U/g)) Antioxidant V550 (Ascorbyl Palmitate) Antioxidant V551 (Tocopherols Mixed (Natural) INCI: Antioxidant Tocopherol) V552 (dl-Alpha Tocopherol (INCI: Antioxidant Tocopherol)) v614 (D-Alpha Tocopherol Natural) Antioxidant S717 (Sodium Disulfite Extra Pure) Antioxidant/preservative P822 (Phycosaccharide AIP) Antioxidant/protective extract M507 (Merquat Plus 3331) Antistatic agent P739 (Polyquarternium-7) Antistatic agent HEW574 (Actiphyte of Witch Hazel PG) Astringent HEW575 (Actiphyte of Horse Chestnut Astringent/anti inflammatory PG) A589 (Organic Aloe Vera Powder 200:1 Botanical extract Freeze Dried) C582 (Codiavelane) Botanical extract C755-LM (Crodarom Velvet Flower) Botanical extract C786-AN (Cosflor Marshmallow HGL-1 Botanical extract (PS)) C787-AN (Cosflor Mango HGL-1 (PS)) Botanical extract E560 (Emblica) Botanical extract HEO503 (Green Tea Lipo S) Botanical extract HEO596 (Organix Marigold PS) Botanical extract HEW1040 (Witch Hazel Organic) Botanical extract HEW1066-MB (Green Tea LG) Botanical extract HEW1085-MB (Ceapro Oat Botanical extract Avenanthramides) HEW1103-MB (Witch Hazel Distilled, Botanical extract Phenoxyethanol) HEW1122 (Specifix Chamomile FG) Botanical extract HEW1130 (Specifix Yarrow 1579) Botanical extract HEW1136 (Specifix Comfrey Leaf PF) Botanical extract HEW1154 (Orange Secrets) Botanical extract HEW503 (Extrait Concombre HG) Botanical extract HEW505 (Aloe Vera 10:1) Botanical extract HEW596 (NAB Fennel Seed Extract) Botanical extract HEW643-LL (Witch Hazel) Botanical extract HEW717 (Extract NAB Red Clover Botanical extract Isoflavones) HEW822 (Green Tea ECO Extract) Botanical extract HEW863 (Rosewater) Botanical extract HEW966 (Calendula ECO) Botanical extract HEW987 (Cosflor Roman Chamomile Botanical extract HGL-1) P850-CC (Pronalen Ruscus Spe) Botanical extract P909 (Proteasyl LS8951 PW) Botanical extract R516 (Regu-SEB (331-01)) Botanical extract R563 (Rosamine R) Botanical extract R566-CC (Remoduline B E1) Botanical extract S981-AN (Slippery Elm Bark Powder) Botanical extract D642 (Dissolvine GL-38) Chelating agent N502 (Dissolvine Na2 (EDTA Na2)) Chelating agent N504 (Dissolvine NA (tetra sodium)) Chelating agent P666-HM (Procircul 8) Circulation active A611 (AC Yeast Beta Glucan) Collagen producing active Z507 (Zinc Oxide EP) colouring agent C766 (Claritea Prov) Dark circle/complexion lightening active S586 (Salicylic Acid) Denaturant P831 (Proteasyl TP POE LS 9818) Elasticity active B515 (Butylene Glycol) Emolient B586 (Cocoa Butter (Blanova)) Emolient C534 (Cetiol OE) Emolient C581 (Cetiol V) Emolient C608 (Organic Cupuacu Butter Refined Emolient Grade) C673 (Cocoa Butter) Emolient C760 (Cetiol C5) Emolient C918 (Cetiol AB) Emolient D585 (Dragoxat 89) Emolient D783 (DC245) Emolient G523 (Glucamate LT) Emolient G549 (Glucamate SSE20) Emolient G550 (Glucate SS) Emolient H568 (Heliogel) Emolient H592-HM (Hexyl Laurate) Emolient H593 (Hydrogenated Polyisobutene) Emolient HEO502 (Aloe Vera Oil Extract CG) Emolient I512 (Isopropyl Palmitate) Emolient I513 (Isopropyl Myristate) Emolient I564 (Isopropyl Myristate) Emolient I569 (Isononyl Isononanoate (Dubb ININ)) Emolient I570 (Isopropyl Isostearate) Emolient L548 (Lipovol MOS-70) Emolient L595 (Linosa PEG-7 Glyceryl Cocoate) Emolient L642 (Lipex Shea) Emolient L645 (Lipex Sheasoft) Emolient M726 (Mikrokil Cos (12002)) Emolient OIL503 (Almond Oil (Sweet) - Refined) Emolient OIL506 (Jojoba Oil Light) Emolient OIL511 (Grapeseed Oil) Emolient OIL512 (Avocado Oil) Emolient OIL520 (Peach Kernel Oil) Emolient OIL522 (Soybean Oil) Emolient OIL533 (Blackcurrant Seed Oil) Emolient OIL536 (Olive Oil - Refined) Emolient OIL541-AN (Castor Oil, Refined) Emolient OIL568 (Organic Jojoba Oil) Emolient OIL572 (Organic Coconut Oil) Emolient OIL574-AN (Hemp Oil, Refined) Emolient OIL590 (Organic Grapeseed Oil) Emolient OIL593 (Calendula Oil 1210) Emolient OIL599 (Organic Shea Butter) Emolient OIL606 (Organic Sweet Almond Oil) Emolient OIL613 (Organic Evening Primrose Oil) Emolient OIL614 (Organic Wheatgerm Oil) Emolient OIL619 (Camelina Oil Refined) Emolient OIL621 (Organic Almond Oil) Emolient OIL633 (Coconut Oil - Fractionated Emolient (liquid)) OIL638 (Rapeseed Oil Refined) Emolient OIL655 (Coconut Oil RD) Emolient OIL658 (Herbal Extract Aloe Vera Oily) Emolient OIL659 (Sunflower Oil Refined) Emolient OIL661 (Jojoba Oil, Golden) Emolient OIL669 (Mineral Oil BO) Emolient OIL676 (Tea Tree Oil Organic) Emolient OIL678 (Olive Oil, Organic) Emolient OIL686 (Avocado Oil Refined) Emolient OIL706 (Organic Castor Oil) Emolient OIL722 (Macadamia Oil Organic) Emolient OIL729-NK (Tamanu Oil, stabilised) Emolient OIL734-CC (Corn Oil Refined) Emolient OIL751 (Hazlenut Oil, Refined) Emolient OIL756 (Rosehip Oil, Refined) Emolient OIL809 (Organic Safflower Oil) Emolient OIL830 (Soyabean Oil Refined) Emolient OIL831 (Lady's Thistle Oil) Emolient OIL841 (Meadowfoam Seet Oil) Emolient OIL853 (Avocado Oil, Crude) Emolient OIL867-AN (Babobab Oil Organic) Emolient OIL870 (Calendula Oil) Emolient OIL907 (Organic Deodorised Argan Oil) Emolient S1014 (Silicone CM56 (DC345)) Emolient S1050 (Saboderm ISN) Emolient S510 (Stearyl Alcohol) Emolient S523 (Silicone DC 200 Fluid 350 CST (see Emolient B665)) S574 (Silicone DC 200/100) Emolient S575 (Silicone DC 1501) Emolient S614 (Silicone DC 245) Emolient V588-AN (Viatenza Shea PO6) Emolient W516 (Wickenol 156 (replaces L503)) Emolient T527 (Tegosoft LSE 65K Soft) Emolient/foam enhancer/viscosity modifier A504 (Amphisol K) Emulsifier A633-AN (Axol C 62 Pellets) Emulsifier C736 (Span 60-PA-(SG) (previoulsy Crill Emulsifier 3 - sorbitan stearate)) C750-CC (Crovol A70 UK) Emulsifier C813 (Crodafos CES) Emulsifier C815 (Ceteareth 25) Emulsifier C933-CC (Cetyl Palmitate 95%) Emulsifier D609 (Dermofeel SL) Emulsifier E529 (Euperlan PK 1200) Emulsifier E633 (Edenor C12 98-100 MY) Emulsifier K570 (Kemest GDS) Emulsifier K571 (Kemionic SLES 228) Emulsifier L536 (Lexemul 561) Emulsifier L583 (Lexemul T) same as G621) Emulsifier L601 (Linosa SLES 70) Emulsifier M519 (Montanov 68) Emulsifier M550 (Montanov 202) Emulsifier M564 (Montanov S) Emulsifier P559 (Procol CS-20D) Emulsifier P680 (Procol CS 20 (Ceteareth-20)) Emulsifier P869-MB (Palmitic Acid) Emulsifier S748 (Surfac JH 200) Emulsifier S840 (Surfhope SE Cosme C-1616) Emulsifier T609 (Tegosoft PC41) Emulsifier S677 (Surfac UN90) Emulsifier/degreasing agent H554-SD (Hydramol TGL Ester) Emulsifier/skin conditioning agent S976 (Sveltine LDRM 961S) Emulsifier/skin conditioning agent S718 (Sorbitan Mono Stearate) Emulsifier/surfactant C810 (Surface CS (Cetearyl Alcohol)) Emulsioin stabiliser B648 (Organic Beeswax) Emulsion stabiliser B661 (Beeswax, white granules (8104)) Emulsion stabiliser C674 (Cetyl Alcohol) Emulsion stabiliser E566 (Emulium Kappa) Emulsion stabiliser M606 (Montanov L) Emulsion stabiliser S1003 (Stearic Acid) Emulsion stabiliser S505 (Stearic Acid 1810) Emulsion stabiliser HEW602 (NAB Willow Bark Extract) Exfoliating BHA E530 (Peeling Enzymatique) Exfoliator F561 (Florabeads 28/60 Arizona Sky) Exfoliator P521 (Pumice Powder Coarse (50 mesh)) Exfoliator T614 (Tabishirex) Exfoliator A607 (Antileukine 6) Extract with UV protection HEW672 (NAB Arnica Extract) Eye active (inflammation/puffiness) L606 (Liponate ISA) Fatty acid L565 (Luviset Clear) Film former L727 (Luviskol K30) Film former P657 (Pepha-Tight) Film former D683 (Daitosol 5500GM K-5136) Film former C759 (Covacryl SP) Film former/conditioning agent C762-SD (Copolymer 845 G) Film former/conditioning agent E531 (Egg White Powder P11) Film former/conditioning agent L713 (Luviquat UltraCare) Film former/hair fixative M663 (Mirustyle XHP) Film former/hair fixative B643 (Biopeptide EL) Firming active T682-CC (Tensine) Firming active T685-CC (Tensine 2) Firming active HEW671 (Extract NAB Mushroom) Firmness and anti wrinkle active S696 (Sodium Bicarbonate) Fizzing agent A640 (GLB60 (Antil HS 60) Foam booster/viscosity modifier D625 (DC949 Cationic Emulsion) Hair conditioning agent M644 (Mirustyle MFP PE) Hair conditioning agent S979-SD (Styleze CC10) Hair conditioning agent T666 (Tinocare SiA1) Hair conditioning agent V603-AN (Varisoft 432 CG) Hair conditioning agent V606-AN (Varisoft BTMS Flake) Hair conditioning agent K558 (Keratec IFP PE) Hair/skin conditioning agent G537 (Glycerin (Surfac G995V)) Humectant G540 (Organic Glycerine) Humectant G628 (Organic Glycerin) Humectant HEW585 (Extrait Ginseng HG) Humectant HEW677 (Extract NAB Siberian Ginseng) Humectant L549 (Lubragel Oil) Humectant L556 (Lactofill Sensitive) Humectant L622-CC (Lubrajel PF) Humectant L717 (Lubrajel Oil Free) Humectant P501 (Propylene Glycol) Humectant P553 (D-Panthenol 751 (liquid)) Humectant P668 (Protachem GL26 (same as L616)) Humectant S521 (Squalane - Olive Derived (was Humectant D527)) S522 (Sodium Hyaluronate 1% Solution Humectant (Liquid)) S545 (Sorbitol Syrup 70% (Sorbidex NC Humectant 16205)) S771 (Sodium Hyaluronate Salt (Powder)) Humectant W503 (Waglinol 6014 (IPM)) Imolient S676 (Solaveil CT-100 Clarus) Inorganic sunscreen Z502 (Zinc Oxide CM3K 50 XZA) Inorganic sunscreen S789 (Suberlift) Lift active N600-CC (Nano LPDs Arbutin PF) Lightening active S952 (Surfac HT10) Low foam surfactant B610 (Bamboo Exfoliator 500) Micro exfoliator D545 (Diamond Powder SY-FS 60/70) Micro exfoliator P601 (Pearl Powder 125) Micro exfoliator R557 (Rice Exfoliator 500) Micro exfoliator I561 (Isocell MAP) Microencapsulated antioxidant U532-CC (UV Titan M 262) Mineral/inorganic sunscreen N598-CC (Norgel) Moisturiser H594-CC (Hydrosoy 2000 PE) Moisturising L547 (Lamesoft PO65) Moisturising V534-CC (Viamerine 4000) Moisturising T557 (Tagravit F1) Moisturising/anti-ageing K562 (Kahl Berry Wax 6290) Natural wax, texturiser A562 (Acusol OP 301) Opacifier M727 (Myristic Acid PC) Opacifier T687 (Tego Pearl N300) Pearliser C510 (Citric Acid Monohydrate) pH adjuster P886 (Potassium Hydroxide) pH adjuster S517 (Sodium Hydroxide) pH adjuster S960 (Sodium Hydroxide 45% solution) pH adjuster T501 (Triethanolamine 99% (T.E.A)) pH adjuster C536 (Calcium Carbonate) pH buffer T691 (Tris Amino Ultra PC) pH buffer G569 (Gelinnov) Polymer L715 (Luvigel EM) Polymer/skin conditioning agent D589 (Dermosoft Octiol) Preservative G609 (Geogard 221) Preservative P590 (Potassium Sorbate Granules 105119) Preservative PR502 (Nipagin M) Preservative PR504 (Kathon CG) Preservative PR505 (Nipasol M) Preservative PR506 (Nipastat) Preservative PR508 (Phenonip) Preservative PR510 (Phenoxetol (Phenoxyethanol) use Preservative PR572) PR515 (Germaben II) Preservative PR518 (Euxyl K100) Preservative PR526 (Euxyl K702) Preservative PR533-CC (Paratexin CPS Preservative (Chlorphenesin)) PR546 (Germall 115 USP (Powder)) Preservative PR547 (Euxyl PE 9010) Preservative PR548 (Grapefruit Seed Extract G2) Preservative PR561 (Optiphen) Preservative PR563 (Benzyl Alcohol) Preservative PR568 (Sodium Benzoate) Preservative PR572 (Saliethanol (phenoxyethanol)) Preservative PR580 (Geogard Ultra) Preservative PR585 (Sorbic Acid) Preservative PR588-LM (Euxyl K712) Preservative PR593 (Euxyl K701) Preservative PR595 (Elestab CPN) Preservative PR607 (BlagGuard GPL (same as PR537)) Preservative S963-CC (Symdiol 68T (177441)) Preservative booster/antioxidant S950 (Sensiva SC 10) Preservative/emolient HEW675 (Actiphyte of White Tea GL) Protective I546 (Protanal FM 6130 (Isagel FM Setting agent Alginate)) A774 (Avenacare ECO Oat Beta Glucan) Skin and hair rejuvenation HEW963 (Oligophycocorail SPE) Skin balancing A734 (Aloe Vera Gel Base) Skin calming HEW885 (Horsetail Extract (1552)) Skin conditioning HEW896 (Camomile ECO) Skin Conditioning A502 (Allantoin 98.9%) Skin Conditioning Agent A676-AN (Aquarich) Skin conditioning agent A732-CC (AC Oak Kernel Protein Powder) Skin conditioning agent B503 (Bernel Ester TCC) Skin conditioning agent C577 (Cosmocair C100) Skin conditioning agent C698 (Cytobiol Lumin Eye) Skin conditioning agent C949-CC (Cetiol S) Skin conditioning agent C954 (Cegesoft C24 (same as T526)) Skin conditioning agent D564 (DC556) Skin conditioning agent D654-AN (Dermofeel P-30) Skin conditioning agent F509 (Fucogel 1000PP) Skin conditioning agent F541 (Fucogel 1.5P) Skin conditioning agent G563 (Gatuline Age Defence 2) Skin Conditioning agent G589-AN (Gluadin Wlm Benz) Skin Conditioning agent G605 (Gatuline Skin Repair Bio) Skin conditioning agent G631 (Gatuline RP) Skin conditioning agent I560 (Isododecane (CSI Code 5108)) Skin conditioning agent K567-AN (Kapilarine) Skin conditioning agent M543 (Mango Butter Refined) Skin conditioning agent M625 (Myritol 312) Skin Conditioning agent P556 (Peg 8) Skin conditioning agent P658 (Pentavitin) Skin Conditioning agent S804 (Shea Butter, Refined) Skin Conditioning agent S825-CC (DC2501 Cosmetic Wax) Skin conditioning agent S982-AN (Sunflohair) Skin Conditioning agent U502 (Urea) Skin conditioning agent V509 (Vitamin F Glycerinester O/S) Skin conditioning agent V546 (Vitamin A (Water Miscible) Type Skin conditioning agent 100) A629-CC (AC Zaatt) Skin conditioning and lightening agent A653 (Achromaxyl IS) Skin lightening agent B677 (Belides ORG) Skin lightening agent P779 (Phytexcell Mulberry) Skin lightening extract D647 (Dismutin J PF) Skin protecting antioxidant S1010 (Schercemol 1688 Ester) Skin/hair conditioning agent T709 (Triethylhexanoin) Skin/hair conditioning agent P839 (Peptan SR Marine) Skin/hair conditioning gent D523-LL (Solubiliser 660352) Solubiliser N608 (Natragem S140NP) Solubiliser P519 (Polysorbate 60 (Protasorb S-20 NF)) Solubiliser P587 (Protasorb O-20) Solubiliser P622-HM (Procol OA-20) Solubiliser P646 (Protachem HCO 40) Solubiliser P880 (PEG-20 Glyceryl Laurate (Tagat Solubiliser L2)) S535 (Surfacare T20) Solubiliser S564 (Solubilisant LRI) Solubiliser C951 (Crillet 3 (Tween 60-SS)) Solubiliser/emulsifier C719-CC (Cibafast H Liquid) Solvent E504 (Ethanol DEB 100) Solvent T619 (Transcutol CG) Solvent HEW980-CC (Cosflor Cucumber HGL-1 Soothing (PS)) P823-HM (Phytexcell Centaury) Soothing/astringent extract A604 (Aquacacteen) Soothing/calming/firming extract HEW673 (Authenticals of Cucumber) Soothing/calming/firming extract S823 (Syntran PC5227CG) SPF booster HEW625 (Actiphyte of Paraguay Tea SPF booster/irritancy reducer of AHA's Conc.) M735 (MALTODEXTRIN (replaces D529)) Stabiliser O564-LL (Oxynex ST Liquid) Sunscreen stabiliser A542 (Steol CA-330-E (AES 136)) Surfactant C777-CC (Crodasinic LS 30) Surfactant D566 (Dehyton K (Sabosol PB)) Surfactant H523 (Hostapon SCI 85 G (Granular)) Surfactant H540 (Hostapon CT TEIG) Surfactant K569 (Kemthox FA S 21 (P776)) Surfactant O523 (Oramix CG 110 (Sue Stowell)) Surfactant O532 (Oramix NS 10) Surfactant P527 (Plantacare 2000 UP (Kemgluco Surfactant CEHL)) P534-AN (Plantacare 818) Surfactant P538 (Kemgluc CLM Plantacare 1200 UP) Surfactant P879 (Procetyl AWS) Surfactant S1047 (Steol CS-330) Surfactant S547-LM (Surfac GMS NSE40) Surfactant S710 (Stepanol AM 30) Surfactant T512 (Tegobetaine F50) Surfactant V599 (Varisoft 300) Surfactant S766 (Steol BES70 D5) Surfactant/emulsifier S777 (Sucragel CF) Surfactant/emulsifier V511 (Varisoft BT85 (Pellets)) Surfactant/hair conditioning agent S795 (Surfacare DHA) Tanning agent E576-HM (Erythrulose) Tanning enhancer C920 (Covabead PMMA) Texturising effect B673 (BRG SG 116 (DC 9040) Texturising Silicone C503 (Carbopol Ultrez 10) Thickener X507 (Xperse 201) UV broad spectrum protection S699 (Sunspheres Powder) UV protection T683 (Tinosorb M) UVA absorber UV504 (Uvinul MS40 (Benzophenone-4)) UVA absorber UV505 (Parsol 1789) UVA absorber UV514 (Uvinul A Plus Granular) UVA absorber Z505 (Z-Cote HP1) UVA and UVB protection UV510 (Eusolex 4360 (Benzophenone-3)) UVA/B absorber E571 (Eusolex t-2000) UVB absorber UV502 (Parsol MCX) UVB absorber UV525-CC (Escalol 587) UVB absorber C955 (Carbopol 980) Viscosity control L721 (Lexfilm Sun) Viscosity control M565 (Mackol CAS 100N) Viscosity control V527-CC (Viscarin GP209NF) Viscosity control A539 (Aculyn 28) Viscosity modifier A601 (Amigel) Viscosity modifier C584 (Carbopol Ultrez 21) Viscosity modifier C594 (Carbopol Ultrez 20) Viscosity modifier H555-HM (Wacker HDK H20) Viscosity Modifier K502 (Keltrol F) Viscosity Modifier K531 (Keltrol CG TE) Viscosity Modifier K532 (Keltrol CG-RD) Viscosity Modifier K537 (Keltrol CG SFT) Viscosity Modifier L651 (Lanette D) Viscosity modifier M528 (Methocel J75 MS) Viscosity Modifier N509 (Natrosol 250 HHR) Viscosity Modifier N533 (Natrosol Plus CS Grade 330) Viscosity Modifier P530 (Pemulen TR 2) Viscosity Modifier S589 (Structure XL) Viscosity modifier S723 (Simulgel NS) Viscosity modifier A554-HM (Antil 171) Viscosity modifier for surfactants S544 (Sepigel 305 4) Viscosity modifier/emulsifier A766 (Avicel PC-611) Viscosity modifiers S1036 (Sylvaclear A200V) Waterproofing S501 (Sodium Chloride (salt)) C905 (Organic Cocoa Butter) Collagen Ingredients Marine Hydrolysed Collagen LMW Hydrolysed Collagen (Marine) Collagen 1% Soluble collagen (porcine) Collagen Hydrolysate Cosmetic N-SS Hydrolysed Collagen Solu-Coll Native Soluble Collagen (Cattle) Solu-Coll M Soluble Collagen (Marine) Solu-Mar Native Soluble Collagen (Marine) Dermosot 1388 Preservative Retinol (tretinoin) Preservative Saponin Preservative Q Enzyme 10 Preservative Glycerrhinitic Acid Preservative Fragrances FRAG1065 (Fresh Style PN994475) FRAG1104 (Celebrity Chic UKB06022) EO806 (Organic Tea Tree Oil) EO807 (Organic Lavender Oil) EO898 (Orange Sweet Essential Oil UN1169)

(38) TABLE-US-00002 TABLE 2 Caprilyc capric triglyceride, Stearic acid, Glyceryl Strearate, Cetearyl Alcohol, Q10, sodium Lauroyl, Glutamate, Retinol Palmitate, Cetearyl Alcohol, Shea butter, Isolanolin, Sorbitan stearate, lactate, urea, Hyluronic Acid, Saponins, D-Panthenol, Glycerin, Sodium Anisate, sodium levulinate, Glyceril Caprilate, EDTA, CaprylyL Glycol, Potassium Sorbate, Caprylic acid combo, Cetyl palmitate, Stearic acid, Cetearyl alcohol, Sodium lauroyl glutamate, Caprilyc capric triglyceride, Sorbitol, Glyceryl stearate, Demin sterile water, Disodium EDTA 0.1%, Hyluronic acid, Retinol (tretinoin) 0.5%, Saponin, Q enzyme10, Glycerrhinitic acid, Collagen, lacto ceramide, L-Cartinine, Lactic acid, Glycolic acid, Sorbitan stearate, Sorbitol, Lactate, Decandenol, Licorice extract, Green tea Extract, Arnica, calcium chloride, gluconate, Thrombin, Demineralised sterile water, Vitamin A, PEG-40 sterarate, Ascorbyl palmitate, Glycine Argenine HCL, Sodium hyluronate, Lauroyl Lysine

(39) TABLE-US-00003 TABLE 3 1 - Bladderwrack extract (seaweed): Derived from the dried thallus (bulbous root) of Fucus vesiculosus, a type of seaweed. 2 - Horsetail extract: Equisetum arvense, commonly known as horsetail, mare's tail, shave grass, or bottle brush, is a plant that grows throughout central Europe. 3 - Hydrocotyl extract: Hydrocotyl asiatica, commonly known as gotu kola or Indian pennywort.

(40) The composition may comprise one or more natural ingredients listed in table 3.

(41) TABLE-US-00004 TABLE 4 EDTA Glycerin Xanthan Gum Avenacare Beta Glucan - Beta Glucan, Water, Maltodextrin Collasurge - Aqua, Collagen Amino Acids, Potassium Sorbate, Ethylhexyl Glycerin, Phenoxyethanol Crotein M - Hydrolysed Collagen Sodium Hydroxide

(42) TABLE-US-00005 TABLE 5 Preservatives: Dermosoft 1388 - Water, Levulinic Acid, Parfum, p-Anisic Acid, Sodium Hydroxide, Glycerin Gerogard 221 - Benzyl alcohol, Water, Dehydroacetic Acid Dermosoft GMCY - Glyceryl Caprylate Potassium sorbate

(43) FIG. 1 shows a side view of an embodiment of a container according to the present invention. The container 10 may be made of any suitable material, for example rigid or flexible plastics.

(44) Alternatively, one of the chambers, 11 may be arranged to be filled with an activator 12a according to the present invention. One of the chambers 13 may be filled with a cosmetically acceptable carrier 19 and may be arranged with an opening, valve or port 18 arranged to allow platelet rich plasma (PRP) to be introduced into the chamber so that it mixes with the cosmetically acceptable carrier. The opening, valve or port may be arranged to be re-sealable after introduction of the PRP, for example by being equipped with a lock or cap or being a rubber seal that can be injected through.

(45) The container may further comprise a mixing chamber 14 in communication with each of the chambers 11 and 13 so arranged that contents of each of the chambers can enter the mixing chamber in a required ratio. A valve, seal, cap or lock may close an exit from the mixing chamber 17 which allows the contents of the mixing chamber to exit the container when required. The container may be activated, for example by squeezing the container or by a mechanism that urges the contents of the two chambers 11 and 13 into the mixing chamber 14 in a required ratio and the mixed contents out of the container via an exit 17. The container may be activated on multiple occasions, each activation causing a suitable portion of the contents of each of the chambers to be mixed together and to exit the container.

(46) FIG. 2a shows a view of an embodiment of an embodiment of the container of the present invention. The container comprises a chamber 20 suitable for containing PRP, the container 20 may be sterile. The container comprises a multiplicity of chambers 21 comprising a cosmetically acceptable carrier 22. The cosmetically acceptable carrier may comprise an activator. The chambers comprising cosmetically acceptable carrier can be opened and a suitable amount of PRP can be added to the cosmetically acceptable carrier and mixed before applying the composition to the skin.

(47) FIG. 2b shows a side view of a chamber suitable for containing PRP. The chamber 23 has a top lid 24 and a bottom lid 25. FIG. 2c shows the chamber 23 with the top lid removed to show that the chamber is equipped with a dropper 26 to allow drops of PRP to be shaken out of the chamber. FIG. 2d shows a view of the chamber 23 upside down with the top lid 24 in place. The bottom lid has been removed. PRP in a syringe 27 with a needle, 28 is injected through a rubber seal 29 that allows PRP to be transferred into the chamber under sterile conditions.

(48) Tests on Human Volunteers

(49) Platelet Rich Plasma is to be used topically, in combination with a Serum containing Collagen. The Collagen in the serum, when in contact with the PRP, induces platelet activation and release growth factors from the activated platelets.

(50) A serum containing collagen has been prepared with the following formulation:

(51) TABLE-US-00006 TABLE 6 CAS Number INCI Function/Description % 7732-18-5 Aqua (Water) Solvent 94.5895 56-81-5 Glycerin Humectant & Moisturiser 2.0015 73049-73-7 Hydrolyzed Collagen Skin Conditioning Agent 1.0600 26402-26-6 Glyceryl Caprylate Emollient, Emulsifier, Emulsifier: HLB 06- 1.0000 10.9 11138-66-2 Xanthan Gum Emulsion Stabiliser, Skin Conditioning 0.9000 Agent, Surfactant, Viscosity Control 110-44-1 Sorbic Acid Antimicrobial 0.3000 139-33-3 Disodium EDTA Chelating Agent 0.1000 9050-36-6 Maltodextrin Absorbant &/or Abrasive Powder, 0.0200 Emulsion Stabiliser, Film Former &/or Hair Fixative 532-32-1 Sodium Benzoate Antimicrobial 0.0100 Oat Beta Glucan Skin Conditioning 0.0100 24634-61-5 590-00-1 Potassium Sorbate Antimicrobial, Vitamin and vitamin 0.0060 derivatives 123-76-2 Levulinic Acid Skin conditioning 0.0015 19856-23-6 Sodium Levulinate Skin conditioning 0.0015 100.0000

(52) Product Specification

(53) The Collagen Serum is a pale yellow, thin liquid with a hazy appearance.

(54) The pH of the Collagen Serum is pH 5.20-5.50 and the Viscosity is 4000-7000 cps using Spindle 4@5.

(55) Product Stability and Preservation

(56) The Collagen Serum remains suitably stable for the duration of a stability test, with a recommended shelf life determined to be 12 months from the date of opening.

(57) The Collagen Serum has passed a full challenge test carried out to the European Pharmacopoeia standards.

(58) Clinical Tests

(59) 9 subjects were recruited who wanted to improve the appearance of areas of their skin. These patients were divided into three groups.

(60) Group 1

(61) Blood was taken from each group 1 subject and centrifuged to separate plasma from other blood components such as red blood cells and white blood cells. Only the richest part of plasma called PRP or F2 fraction was collected as this is the fraction that is richest in platelets. The poorer fraction: PPP or F1 was not used in the serum.

(62) PRP was injected into the skin using a series of intradermal injections across the treated area. The remaining PRP was stored. Each day for the following eight days a sample of PRP was mixed with equal volume of serum containing collagen and the mixture was applied to the treated area. On day eight further blood was taken and fresh PRP F2 was collected so that the topical treatment was continued for another 8 days after the day of injection treatment.

(63) Group 2

(64) Blood was taken from each group 2 subject and centrifuged to separate plasma from other blood components such as red blood cells and white blood cells. Only the richest part of the plasma, PRP fraction or F2 was separated and collected from the rest of the plasma as this is the fraction that is richest in platelets.

(65) The PRP was stored. Each day for the following eight days a sample of PRP was mixed with equal volume of serum containing collagen and the mixture was applied to the treated area on one side of the subject's face. On day 8 and 16 further blood was taken and fresh PRP (or F2) was collected so that the topical treatment was continued for a total of 24 days after the start of treatment treatment. No PRP injections were given to this group, only topical treatment with PRP mixed with collagen serum.

(66) Group 3

(67) The subjects in group 2 applied the collagen serum twice a day for 24 days without any injections or any PRP mixed with the serum.

(68) Results

(69) The appearance of the skin on the treated area before and after the treatment for each subject is shown in FIG. 4. Pictures of the treated areas of patients 1, 2 and 3 from group 1 and patients 1, 2 and 3 from group 2 show a comparison of the skin before the treatment and after the treatment. Group 1 had PRP injections in the treated area followed by using serum mixed with PRP each day for 16 days. Group 2only used serum mixed with PRP each day for 24 days on one side of their face so we could compare the difference with the other side of their face Group 3used the collagen serum only (without adding PRP to it).

(70) After using the PRP Injections for as anti aging treatment we know that PRP activates tissue regeneration and improves the tone and skin elasticity.

(71) By: stimulating the natural production of Hyaluronic Acid by our own cells promoting the increased secretion of Collagen and Elastin which generates a greater consistency, firmness and reduced sagging in the skin it improves the skin hydration softens lines and wrinkles it increases the luminosity and gives the skin a higher luster.

(72) The only problem with the injections is the post operative swelling, bruising caused by many injections all over the sensitive areas like face and neck, soreness and the fact that the effect doesn't last very long which is why the present invention will dramatically improve the effect of the PRP, here dude the recovery time from the post operative problems and maintain the results for much longer. The only problem with the injections is the post operative swelling, bruising caused by many injections all over the sensitive areas like face and neck, soreness and the fact that the effect doesn't last very long. The advantage of the present invention is that mixing PRP with serum and using it post PRP injections dramatically improves and prolongs the effect of the PRP and also allows the active factors to penetrate into the skin without the need for injection. The PRP with serum can be used as a course of treatment where a portion of PRP is mixed with a portion of serum each day to activate fresh platelets every day and the activated platelets are applied to the skin. This treatment can be carried out alone or after a course of treatment of PRP injection to prolong and enhance the effects of the PRP by injection treatment and also to help reduce the amount of bruising, swelling and soreness caused by the PRP by injection treatment.

(73) Feed Back from the Clients in Group 1, 2 and 3

(74) Group 1

(75) Client 1: was a 47 years old lady who had PRP injections both in the face and hands followed by using our plasma serum for 8 days. Before the treatment she suffered from red patchy areas on her cheeks and the side of her nose as you can see in the first picture below on the left. Immediately after the PRP injections her face was slightly swollen and inflamed because of the injections. It also felt very sore. I made her the plasma serum that day and she started using it immediately and carried on for 8 days.

(76) After 8 days the red patchy areas were all gone. The dark circles under the eyes improved. She reported that her skin felt softer, plumper and more hydrated and she had a healthy Glow. She no longer needed to use any foundation on her skin.

(77) The same client had her hands treated by PRP injections followed by 8 days using the plasma serum

(78) Hands Before:

(79) Skin was very dry. Red patchy areas and deep lines around the wrist. Even using hand creams several times daily was not enough to keep the skin moisturized and soft. As a result skin looked old and wrinkly

(80) Hands after Treatment

(81) The skin was much softer, tighter and more hydrated. The redness had faded dramatically on the wrist and the deep lines had softened.

(82) The soreness and bruising after the PRP injections healed very quickly, much quicker than it would normally

(83) The skin on the hands felt soft and was glowing. It was softer, more hydrated, lines and patchy red areas had faded dramatically

(84) Client 2: This was a 67 years old lady with deep frown lines and lots of smaller lines around the eyes, on the cheeks and the chin. She had deep dark circles below the eyes and patchy red/brown areas all over her face. To this lady sagging was the biggest problem and previous filler treatments on her face weren't giving her the result she desired.

(85) She was treated with just PRP injections 9 months ago. She took about 5 days to recover from post-operative swelling and bruising. After that skin felt great but it didn't last very long.

(86) This time I treated her with PRP injections first followed by using my Plasma serum for 8 days.

(87) Not only she recovered from post-operative swelling, soreness and bruising within 24 hours, her skin continued to feel softer, plumper, and brighter and had a very healthy glow. The biggest improvement with this lady was the tightening of the skin. She described it as A gentle face lift

(88) The little lines on the cheeks and chin softened dramatically. The deep frown lines also softened. The areas under her eyes looked brighter and healthier and her skin had a more even and brighter tone.

(89) Client 3 This was a 32 years old man with a very deep Nasio Labial fold (laughter line) which made him look older than his age. The skin inside the fold was also very dry and made shaving very hard for him.

(90) This subject was treated with one session of deep PRP injections followed by using the Plasma serum for 8 days. The fold didn't completely disappear but improved a lot, it now looks more like a fine line. The skin also felt softer, tighter and more hydrated.

(91) Group 2:

(92) The clients in this group reported very similar feedback: The treated side of the face felt much tighter, more hydrated, plumper and smoother than the untreated side throughout the whole 24 days despite being out in the sun and wind.

(93) The dark circle around the eyes looked much lighter.

(94) They all reported a new healthy Glow

(95) As a result of this rehydration the lines around the eyes looked much softer.

(96) Those who had very dry and patchy skin and after using the serum for 24 days reported softer, plumper, tighter and more even tone on the skin on the treated side while the untreated side remained the same.

(97) GROUP 3: This Group Reported No Significant or Visible Improvements Other than the skin felt a little more moisturized whenever they applied the collagen serum on but the effect didn't last more than 2-3 hours.

(98) Kit

(99) The kit may contain:

(100) Eight plastic capsules containing the collagen serum (cosmetically acceptable carrier comprising collagen activator) for 8 days use or a suitable number of plastic capsules, one for each day or one for each application of the PRP. The amount of collagen serum in each capsule may be enough for one application. The collagen serum will be mixed with the same volume of PRP, mixed between finger tips and applied on the face, neck, chest and/or hands.

(101) A container for holding the PRP made of a suitable material, for example Steriline polypropylene which according to the test described in this application has proven to be the best material for storage of PRP (also called F2 which is the richer part of plasma).

(102) The PRP container may be designed to reduce the amount of air that flows into the bottle so that there is some oxygen inside but not a constant flow of oxygen. The container may be arranged to dispense the PRP in a dropwise manner so that the user can dispense a suitable number of drops of PRP to be mixed with the cosmetically acceptable carrier for each application.

(103) 3 A small battery driven vibrating table is also included now so that the client can keep the PRP agitated for the 8 days. Although our test showed great results even with the PRP group that was only shaken a few times/day we know that the best results are always achieved if the PRP is agitated constantly which is why a little vibrating table is in the kit.

(104) Platelet Storage Conditions

(105) Whilst PRP is generally preferred to recombinant growth factors in promotion of wound healing there is a paucity of data on stability of PRP preparations in this clinical setting. Being anucleate, platelets have a short in vivo lifespan of 8-10 days, and platelet donations taken into citrate-based anticoagulants stored at 22 C.2 C. with continuous, gentle agitation are generally assigned a 5 day expiry. In our study we investigated serial (daily), collagen-induced PDGF-BB release from normal donor platelets stored at room temperature over 8 days under variable conditions.

(106) Collection of Donor Platelets

(107) A total of 125.0 mL of blood was taken from a haemostatically asymptomatic, clinically well adult male donor into 25 BD Vacutainer tubes (Bunzl Healthcare, Enfield, UK), each containing 1.0 mL of Acid-Citrate-Dextrose solution B (ACD-B) as the anticoagulant.

(108) The non-traumatic venepuncture was performed with minimal stasis using a 21 g-butterfly needle. The donor had not received any drugs known to affect platelet function in the preceding two weeks, or any foodstuffs known to affect platelet function in the preceding five days.

(109) Blood for platelet diagnostics is normally taken into 0.105M tri-sodium citrate in a ratio of nine parts blood to one part anticoagulant. However, this is based on diagnostic assays being performed within four hours of venepuncture to assess function on freshly drawn platelets before natural deterioration adversely affects diagnostic accuracy. The choice of ACD-B as anticoagulant for this study was based on the work of Lei et al who demonstrated that ACD-B was superior to tri-sodium citrate in maintaining platelet structure integrity and preventing spontaneous aggregation.

(110) Preparation of Platelet-Rich Plasma

(111) The blood was allowed to cool for 15 minutes after the venepuncture to prevent formation of plasma clots. The tubes were then centrifuged at 22 C. in an IEC Centra CL3 centrifuge (Thermo Scientific, Basingstoke, UK) at 130 RCF (800 RPM) for 20 minutes to generate PRP.

(112) After centrifugation, the PRP was pooled by transferring into two 30 mL Sterilin polypropylene universal containers (Thermo Scientific) using plastic transfer pipettes and avoiding the white blood cell-rich buffy coat layer. Use of polypropylene containers and plastic pipettes prevents platelet activation during sample manipulation. The contents of the two containers were multiply inter-mixed to achieve homogeneity.

(113) The remaining whole blood was centrifuged at 2450 RCF (3500 RPM) for 10 minutes to obtain platelet poor plasma (PPP) for use as a blank in the collagen-induced platelet activation. The PPP was transferred into a separate universal container.

(114) A blood count was performed on the PRP [12] using a Sysmex PocH 100i (Sysmex UK, Milton Keynes, UK) to check that:

(115) The platelet count was between 150-600109/L

(116) The white blood cell count was <0.5109/L

(117) The red blood cell count was <0.51012/L

(118) If the platelet count is too high, artefactual inhibition of in vitro aggregation can occur, and too many red blood cells will interfere with detection of aggregation. White blood cells can inhibit platelet aggregation so it is important that most of them are removed from PRP.

(119) Collagen-Activated Platelet Aggregometry

(120) Immediately after preparation, aliquots of the PRP were subjected to activation by Collagen Reagent HORM (Takeda Austria, Linz, Austria), an equine tendon collagen suspension, in a PAP8 platelet aggregometer (Alpha Laboratories, Eastleigh, UK). The PAP8 employs light transmittance aggregometry, which is summarised in FIG. 5.

(121) The platelets were activated with collagen at a final concentration in the PRP of 5 g/mL, a standard concentration for diagnostic purposes that should instigate full aggregation in normal platelets. Separate aliquots were activated with collagen at a concentration of 10 g/mL to ascertain whether doubling the dose might maximise aggregation, and thus, -granule content release. Once aggregation was complete, the aliquots were centrifuged to pellet the platelet aggregates and the supernatant removed and frozen at 80 C.

(122) Storage of Platelet-Rich Plasma

(123) The remaining PRP was stored under different conditions in two types of container. Container 1 was a 70 mL Nunc non-treated flask (Thermo Fisher Scientific, Langenselbold, Germany), a sterile, non-activating polystyrene tissue culture flask, which is shown in FIG. 6(a). Although the polystyrene body of the flask is not oxygen permeable, the filter cap permits a constant air flow. Container 2 was a 30 mL Sterilin polypropylene universal container (Thermo Scientific), which is sterile but not oxygen permeable, which is shown in FIG. 6(b).

(124) Each container type was used to store PRP in four different ways over an eight day period, as described below: (a) Kept agitated throughout an 8 day storage (b) Gently shaken/inverted several times per day (c) Kept stationary throughout (d) Kept agitated throughout an 8 day storage+addition of PGE1

(125) The 150 mL volume of donated blood plus anticoagulant yielded approximately 65 mL of PRP, which permitted storage of approximately 8.0 mL of PRP in each container.

(126) It has long been recognised that gently agitating platelets during storage reduces activation and debris formation. It has been shown that an interruption of one day has negligible, measurable effect, but longer periods can result in significant, deleterious changes. For this reason, aliquots of PRP were stored constantly agitated, intermittently agitated/mixed or kept stationary to assess the impact of variable agitation. An additional constantly agitated storage condition was introduced to include addition of prostaglandin E1 (PGE1), a natural platelet inhibitor that triggers an increase in cyclic adenosine monophosphate levels which counteracts platelet activation by reducing calcium flux. Addition of PGE1 to PRP reduces platelet activation during storage whilst permitting a response to most agonists during in vitro analysis. PGE1 was added to the PRP at a final concentration of 10 g/mL, the standard concentration for diagnostic use. All PRP was stored at room temperature since cold storage activates platelets.

(127) A Luckham R100 Rotatest Shaker (FIG. 7) was used to agitate the containers requiring constant agitation. The platform was rotated on setting 5, a mid-point setting to approximately mimic that employed for platelets being stored for transfusion. The aliquots being mixed intermittently were shaken/inverted hourly during the laboratory core hours of 09.00-17.00 h for the first five days and left undisturbed for days six and seven as they were over a weekend.

(128) Serial Collagen-Activated Platelet Aggregometry

(129) At approximately the same time each day as when the freshly prepared PRP was analysed, aliquots of mixed PRP from each of the eight containers were separately activated in the PAP8 aggregometer with collagen at a final concentration in PRP of 10 g/mL and the aggregation patterns recorded. Once aggregation was complete, the aliquots were centrifuged to pellet the platelet aggregates and the supernatants removed and frozen at 80 C. This was undertaken on days 2-5 and day 8. Platelet counts were also performed each day as an on-going assessment of PRP quality and platelet activation and spontaneous aggregation. Platelet counts were performed immediately prior to aggregometry.

(130) Analysis of Supernatants for PDGF-BB

(131) To assess storage and release of growth factors from stored platelets, measurement of PDGF-BB was chosen as a representative marker since it has been employed in similar studies for the same purpose, becaplermin is a recombinant version of PDGF-BB, and reagents for analysing PDGF-BB levels are commercially available.

(132) PDGF-BB levels in the supernatants were quantified with Human PDGF-BB Platinum ELISA reagent kit (Affymetrix eBioscience, Hatfield, UK). The principle of measurement of PDGF-BB by enzyme-linked immunosorbent assay (ELISA) is depicted in FIG. 8.

(133) Prior to use in quantification of released PDGF-BB in the stored supernatants, the kit was evaluated for standard curve linearity and analytical precision. Whilst each step of the assay was performed manually, end-point detection, standard curve generation, test raw data and calculation of final results were all performed on a Dynex DS2 ELISA analyser (Instrumentation Laboratory, Warrington, UK).

(134) Cell Counting of PRP on the Day of Donation

(135) The blood cell counts on the PRP immediately after harvesting, and prior to collagen-induced platelet aggregation, are shown in Table 1.

(136) TABLE-US-00007 TABLE 7 Cell counts on PRP Parameter Results Units White cell count 0.0 109/L Red cell count 0.00 1012/L Platelet count 236 109/L

(137) The aliquots activated with 5 g/mL collagen achieved 93% and 85% aggregation (mean 89%). The aliquots activated with 10 g/mL collagen achieved 91% and 84% aggregation (mean 88%). Although this revealed no increase in the activation/aggregation responses to a doubling of the standard collagen concentration, the 10 g/mL collagen concentration was adopted for the serial testing to allow for ageing of the platelets during the course of the experiments.

(138) Collagen-Induced Aggregation of Stored PRP: Day 2

(139) Collagen-induced aggregometry was performed in duplicate on aliquots of PRP from each storage situation. The aggregation traces for PRP stored in the Nunc flasks are shown in FIG. 10, and the non-oxygen permeable universal containers in FIG. 11. Platelet count (Plt) prior to aggregometry and mean percentage final aggregation (MA) for each duplicate are given in the legends.

(140) Collagen-Induced Aggregation of Stored PRP: Day 3

(141) Collagen-induced aggregometry was performed in duplicate on aliquots of PRP from each storage situation. The aggregation traces for PRP stored in the Nunc flasks are shown in FIG. 12, and the non-oxygen permeable universal containers in FIG. 13.

(142) Platelet count (Plt) prior to aggregometry and mean percentage final aggregation (MA) for each duplicate are given in the legends

(143) Collagen-Induced Aggregation of Stored PRP: Day 4

(144) Collagen-induced aggregometry was performed in duplicate on aliquots of PRP from each storage situation. The aggregation traces for PRP stored in the Nunc flasks are shown in FIG. 14, and the non-oxygen permeable universal containers in FIG. 15 Platelet count (Plt) prior to aggregometry and mean percentage final aggregation (MA) for each duplicate are given in the legends.

(145) Collagen-Induced Aggregation of Stored PRP: Day 8

(146) Collagen-induced aggregometry was performed in duplicate on aliquots of PRP from each storage situation. The aggregation traces for PRP stored in the Nunc flasks are shown in FIG. 18, and the non-oxygen permeable universal containers in FIG. 19 Platelet count (Plt) prior to aggregometry and mean percentage final aggregation (MA) for each duplicate are given in the legends.

(147) Plots of changes in % aggregation over time in variably stored PRP are shown in FIGS. 20-23.

(148) Platelet Parameters Over Time in Variably Stored PRP

(149) In addition to counting platelet numbers, the Sysmex PocH 100i analyser generates other platelet diagnostic parameters, as described below:

(150) Mean platelet volume (MPV)

(151) Platelet distribution width (PDW) calculates the relative width of platelet volume distribution

(152) Platelet-large cell ratio (P-LCR) is the ratio of platelets smaller than 12 fL to those with a volume between 12-30 fL

(153) Reference ranges from literature for whole blood are as follows: MPV: 7.5-11.5 fL PDW: 9.3-16.0 fL P-LCR: 24.8-41.2%

(154) Table 8 shows the platelet parameters in variably stored PRP immediately prior to collagen activation on each day.

(155) TABLE-US-00008 TABLE 8 Serial platelet parameters in variably stored PRP Storage Storage Platelet Day of analysis container conditions parameters Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 No storage Fresh PRP PDW (fL) 11.6 MPV (fL) 9.7 P-LCR (%) 22.3 Nunc flask Constant PDW (fL) 16.2 15.2 14.0 12.2 8.8 agitation MPV (fL) 11.8 11.1 10.7 10.1 8.6 P-LCR (%) 41.2 36.0 33.0 28.5 18.4 Intermittent PDW (fL) 13.4 17.5 14.4 14.2 12.1 agitation MPV (fL) 10.7 11.8 11.3 11.0 10.4 P-LCR (%) 29.7 41.8 36.4 34.7 30.5 Stationary PDW (fL) 12.5 16.4 16.1 15.2 13.9 MPV (fL) 10.2 11.7 11.7 11.4 11.0 P-LCR (%) 26.4 41.5 41.2 38.0 35.6 Constant PDW (fL) 16.5 15.2 14.1 12.2 9.7 agitation + PGE.sub.1 MPV (fL) 11.7 11.1 10.9 10.2 8.9 P-LCR (%) 41.3 36.4 34.4 29.1 20.2 Universal Constant PDW (fL) 9.9 9.4 No 8.6 8.3 agitation MPV (fL) 8.8 8.6 analyser 8.6 8.4 P-LCR (%) 16.9 17.9 output 19.2 18.3 Intermittent PDW (fL) 11.3 11.6 12.6 12.0 13.8 agitation MPV (fL) 9.6 9.7 10.2 10.1 10.7 P-LCR (%) 21.0 21.9 25.8 24.9 31.1 Stationary PDW (fL) 11.0 11.8 11.5 12.3 14.4 MPV (fL) 9.5 10.1 10.1 10.1 11.4 P-LCR (%) 19.6 23.8 23.8 23.5 36.6 Constant PDW (fL) 9.8 8.7 8.5 7.8 8.9 agitation + PGE.sub.1 MPV (fL) 8.8 8.3 8.1 8.1 8.6 P-LCR (%) 16.4 17.1 13.8 15.4 20.2

(156) TABLE-US-00009 TABLE 9 Serial PDGF-BB levels in variably stored collagen (10 g/ml) activated platelets Storage Storage PDGF-BB (pg/mL) container conditions Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 No storage Fresh PRP 18 734 Nunc flask Constant 13 190 14 580 11 081 13 689 10 332 agitation Intermittent 12 871 11 488 10 589 9 991 10 042 agitation Stationary 11 159 12 441 10 973 11 969 11 210 Constant Not 10 055 7 842 9 796 9 808 agitation + PGE.sub.2 available Universal Constant Not 12 486 12 905 10 950 9 925 agitation available Intermittent 15 303 14 141 12 514 10 547 8 701 agitation Stationary 10 875 13 649 12 939 10 938 11 398 Constant 13 082 11 836 9 829 10 148 8 967 agitation + PGE.sub.3 Note that results for Day 2 of constant agitation + PGE.sub.1 in the Nunc flask and constant agitation in the universal are unavailable due to technical problems

(157) Virtually without exception, publications on the use of Plasma for cosmetic and wound healing uses describe use of freshly drawn PRP, or in some cases, PRP that has been frozen and thawed. The aim of the present study was to evaluate growth factor release from PRP stored at room temperature over a number of days, using PDGF-BB as the marker. Freezing PRP is known to damage the platelets causing a rapid release of growth factors on thawing. For this reason frozen PRP may not be useful for techniques where the plasma is stored for use on several consecutive days unless the PRP is frozen in portions so that one may be thawed immediately before each application.

(158) Effects of Storage on Platelet Number and Size Parameters

(159) There was a steady fall in platelet count over time in all storage containers and conditions except for the two universals under constant agitation which exhibited a marked fall on Day 2 before beginning a steady decline after that. There is no immediately obvious explanation for this observation, although the platelet parameter results are revealing. Both these containers had lower PDW, MPV and P-LCR values than the stationary and intermittently agitated universals on all days of analysis. The smaller platelet size, narrower width distribution and lower ratio suggest that larger platelets were missing from these two containers, but why this may be the case is unclear. It would be tempting to suggest that the intensity of agitation was too high and had achieved a degree of activation, with the more reactive larger platelets perhaps being more susceptible, yet this was not mirrored in the two constantly agitated Nunc flasks with apparently greater oxygen access. The otherwise steady decline in platelet count in all containers will have been largely due to the well described platelet storage lesion.

(160) Remarkably, all PDW, MPV and P-LCR results from universal-stored PRP were lower than their Nunc flask-stored counterparts, indicating container-induced effects. Furthermore, platelet parameters on Day 2 were closer to those of fresh PRP in the universal-stored PRP than Nunc flask-stored PRP. Minor increases in MPV and PDW during storage up to five days have been reported previously but not to the marked extent of the PDW results in the Nunc flask-stored PRP. If MPV does not normally increase significantly over this time in stored PRP, the moderate increase on Day 2 in these flasks accompanied by marked increase in PDW, particularly in the constantly agitated flasks, is suggestive that a degree of microaggregate formation had occurred. This would explain the increase in P-LCR that was suggesting the presence of higher numbers of larger cells, which were more likely to have been microaggregates. The only moderately increased MPVs indicate they remained small enough for the vast majority to be counted as platelets. Interestingly, PDW, MPV and P-LCR reduced over time in the constantly agitated Nunc flasks, probably due to increasing spontaneous aggregation over time such that the largest aggregates were not aspirated into the blood count analyser. Further evidence of microaggregate formation was found from apparent low red blood cell counts in many of the Nunc flask-stored PRP results (data not shown). There were no red cells in the PRP on Day 1 and these counts were due to small populations of aggregates too large to be counted as platelets. The only universal-stored PRP to register a low red cell count was from the stationary universal on Day 8, which had the highest PDW, MPV and P-LCR values of any universal-stored PRP. MPV was more stable over time in the intermittently agitated and stationary Nunc flasks, suggesting that constant agitation exacerbated spontaneous aggregate formation in a storage situation where low-level activation was already more likely. There was little difference between the platelet parameters of the constantly agitated Nunc flasks with and without PGE1, or the universals, so it appears that the PGE1 did not suppress spontaneous aggregation. It may be that a higher concentration was required, or that the PGE1 is insufficiently stable for stored PRP.

(161) Converse results were seen in universal-stored PRP in that PDW, MPV and P-LCR were lower in the constantly agitated universals than in the intermittently agitated and stationary universals. As described above, there was a curious fall in platelet count, PDW, MPV and P-LCR parameters on Day 2 in the constantly agitated universals, which may have been due to an immediate, more intense formation of aggregates that were not counted in the analyser, followed by relatively small reductions in PDW and MPV over time. In contrast to the steep falls in P-LCR over time in the constantly agitated Nunc flasks, the counterpart universals exhibited mild increase over time, suggesting low level aggregate formation. Some degree of activation and aggregate formation is expected in stored platelets. Only the intermittently agitated and stationary universals mirrored baseline PDW, MPV and P-LCR values on Day 2, with the anticipated gradual but small increases over time of PDW and MPV. The increases in P-LCR over time were more marked, suggesting a degree of aggregate formation in these containers too.

(162) Based on these observations, the intermittently agitated universal maintained the greater platelet integrity over time. Hunter et al showed that interruption of agitation for one day produces no measurable platelet damage, so the intermittent agitation during laboratory core hours and less than 24 hour interruption overnight appears to have been sufficient to maintain comparable integrity.

(163) Effects of Storage on Platelet Aggregation Responses

(164) As previously reported, there was an overall continuous fall in platelet aggregability over time under all storage conditions in the study. An unanticipated finding was the remarkable fall in final percentage aggregation of Nunc flask-stored PRP on Day 2 and thereafter, in stark contrast to the more gradual declines in universal-stored PRP. The minimal platelet count reductions, other than from the constantly agitated universals on Day 2, indicate that relatively few platelets had formed aggregates and that the aggregation responses were a result of activation of the majority of the stored platelets. In which case, the storage conditions in the Nunc flasks were detrimental to overall platelet function.

(165) Polypropylene is the preferred material for platelet storage although platelet adhesion to untreated polystyrene is normally minimal with non-activated platelets. However, binding of plasma proteins such as fibrinogen and VWF to the polystyrene can promote platelet adhesion and subsequent activation. Again, the minimal fall in platelet counts suggest this either did not occur, or occurred only minimally. Thus, the container itself would not be expected to inhibit platelet function and conditions in the PRP itself were the more likely culprit. The most probable explanation for such marked storage-induced differences in initially identical samples of PRP is a drift in plasma pH. Watts et al showed that PRP stored in a closed system to maintain pH better preserved platelet function than PRP stored in a controlled CO2/air environment, which was probably related to the presence of an air/liquid interface in the latter. The universals have screw top lids which were only removed once a day to extract PRP for aggregometry, whilst the Nunc flasks permitted air flow. Since the 30 mL universals were used to store only approximately 8.0 mL of PRP, they likely retained sufficient oxygen in the dead space until the next opening to not significantly impair platelet integrity. The anticoagulant used for sample collection can also affect pH of PRP.

(166) A commonality in aggregometry responses between the two storage containers is the reduced final percentage aggregation in PRP from the constantly agitated samples compared to those intermittently agitated or kept stationary. This is counter-intuitive as accepted dogma is that stored platelets should be constantly, gently agitated to reduce activation and debris formation. It may be that the agitation was too harsh or too gentle for the PRP volumes and containers that were employed, or that pH changes were exaggerated by the constant movement, even in the universals since they retained an appreciable amount of air.

(167) Effects of Storage on PDGF-BB Levels

(168) The amount of released PDGF-BB in freshly prepared PRP is affected by numerous variables, including anticoagulant, donor whole blood platelet count, PRP preparation conditions, platelet count in PRP and type and concentration of agonist used to induce release. Nonetheless, the PDGF-BB level of 18 734 pg/mL in the freshly prepared PRP in this study broadly mapped to levels reported by other workers. From similar centrifugation conditions, Gonzalez et al reported a PDGF-BB range of 9.6-14.6 ng/mL from PRP obtained from 32 donors, and Castillo et al reported 13.0-33.2 ng/mL from five donors.

(169) A marked fall in PDGF-BB levels was observed on Day 2 in all storage conditions where results were available. The mean percentage reduction was 32%, ranging from the 18% reduction in the intermittently agitated universal to the 42% reduction in the stationary universal. Most were followed by a steady, continuous decrease in PDGF-BB levels, some ostensibly reaching a plateau by Days 5 and 8. To a large extent these findings are unsurprising as platelet secretory capacity falls during storage, yet by Day 8, appreciable levels of PDGF-BB remained despite the expectation that the majority of platelets would be dead or poorly functional by that time. A potential explanation is that the PDGF-BB in the supernatant may have merely leaked from the platelets as they aged and membrane integrity was lost. In view of the minimal reduction in platelet counts for most storage conditions, and even the steady fall after the initial marked reduction in the constantly agitated universals, this would seem unlikely to be the only causative factor. Furthermore, if the platelets were fragmenting over time there would likely have been gradual reductions in MPV, which was only observed in constantly agitated Nunc flasks, whilst others remained stable or increased over time. However, microaggregate formation could mask more subtle volume loss from membrane fragmentation. If those MPV reductions in the constantly agitated Nunc flasks were due to fragmentation, a concomitant increase in PDW over time would be expected, but the converse was the case.

(170) What is also interesting is that the relatively poor aggregation responses from Day 2 onwards in constantly agitated Nunc flask-stored PRP are not accompanied by similarly marked PDGF-BB reductions. This was true, albeit to a lesser extent, with the other paired storage conditions. It appears that some of the PDGF-BB in the supernatants is not derived from platelet release, and the explanation for this comes from other studies that additionally measured PDGF-BB in PPP. Gonzalez et al reported a range in PPP of 8.8-12.6 ng/mL, whilst Valeri et al reported a range of 1549-2813 pg/mL. There are clear between-study differences yet plasma PDGF-BB does contribute to values measured in platelet releasates unless platelets are washed prior to analysis. The mechanical trauma of PPP and PRP preparation will also have contributed to the presence of free PDGF-BB in the PPP. The platelet counts in the PRP of the study by Gonzalez et al were adjusted to 250109/L, similar to the count in the donor PRP for this study, whilst the mean count in PRPs from the study by Castillo et al was 566109/L. From a clinical perspective, the PDGF-BB values from collagen-activated PRP reported in this study relate to a lower platelet count than would be used in clinical practice, so the differentiation between release from fresh and stored PRP would be greater. Again, the aim of the study was to investigate relative loss of PDGF-BB release, which had to be performed at lower platelet counts to maximise application of available analytical techniques.

(171) Parameter Stability

(172) Platelet rich plasma stored in the intermittently agitated universal maintained better stability in the parameters studied than that stored in other conditions. Up to Day 5 there was minimal reduction in platelet count and elevations in PDW, MPV and P-LCR, particularly in comparison with some of the marked platelet parameter changes in PRP stored in Nunc flasks. The platelet changes in the stationary universal were greater except the platelet count, and there is no clear explanation for the marked fall in platelet count on Day 2 in the two constantly agitated universals.

(173) Platelet aggregation responses to collagen activation were lower in Nunc flask-stored PRP than universal stored PRP for the reasons described above. Higher final percentage aggregation over time was achieved by the PRP in intermittently agitated and stationary universals compared to their constantly agitated partners, other than an apparent surge on Days 5 and 8 in the universal without PGE1. However, the falls in aggregation (representing overall platelet reactivity), particularly in Nunc flask-stored PRP, were not accompanied by similarly dramatic reductions in supernatant PDGF-BB levels. It seems likely that although secretory capacity reduced over time, PDGF-BB levels did not fall to near zero due to a mixture of leakage from senescing platelets, mechanical trauma and innate plasma levels.

(174) Although PRP stored in the intermittently agitated universal maintained better overall stability, it did in fact generate the lowest PDGF-BB value on Day 8 despite achieving 39% final aggregation. An important consideration is that the study merely assessed PDGF-BB concentration but not function, so it is impossible to know what levels of PDGF-BB are therapeutically efficacious, and therefore, at what point during storage the PRP should be discarded. Furthermore, the PDGF-BB itself may lose functionality over time and be therapeutically ineffective despite an apparently high level of release.

(175) Consideration of function aside, PDGF-BB levels from activation of PRP under all storage conditions were within the range reported by Gonzlez et al for fresh PRP with a similar platelet count, up to Day 5. Although this may be encouraging in terms of storage potential, all the values were markedly lower than for fresh PRP. Without knowledge of PDGF-BB functionality, or that of other released growth factors, it would seem that freezing either the fresh PRP or the post-activation supernatant might generate a more efficacious therapeutic product. Whilst acknowledging the controversy regarding PRP cryopreservation, Roffi et al showed that frozen-thawed PRP without prior agonist activation achieved comparable in vitro effects on cultured cells to those of fresh PRP despite reductions in PDGF-AB/BB levels. The freeze/thaw cycle acts as a surrogate activator by disrupting platelet membranes and inducing content release. In view of the lower growth factor levels in the freeze/thawed PRP, it may be a less efficient approach to increasing growth factor availability than agonist-induced platelet activation since they are packaged within -granules rather than free in the cytoplasm. Durante et al reported PDGF-BB levels in fresh PRP of 17 9041346 pg/mL but reduced levels even after three freeze/thaw cycles. This suggests that freezing the post-collagen activation supernatant is more likely to generate and preserve higher growth factor levels, with the possible additional value of excluding platelet debris from the final product.