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Treatment of idiopathic macular rupture with platelet-rich blood plasma

 A promising direction in IMR surgery is the use of BOTP blood, which is plasma obtained from autologous human blood with a platelet concentration of about 1 mln cells/μl.


Platelet-rich blood plasma (rationale of the method)


 Many terms are used in the literature to refer to BOTP. The most common among them is "platelet concentrate". This is not quite correct, because a true platelet concentrate must contain only platelets, without plasma, and therefore cannot clot and form a clot. In clinical practice, a product that is a concentrate of platelets in a small volume of plasma is used, and therefore it would be more correct to call it "platelet-rich plasma. The term "platelet gel" is also not accurate. BOTP is nothing more than a blood clot in which there is an increased concentration of platelets. This clot contains adhesive molecules that enhance the biological activity of the clot, while there are none in the gel.


Platelets are of key importance as an intermediate link in the process of healing the damaged tissue. In a healthy person, the number of platelets in blood varies from 150-450 x 109/l. A large number of adhesive receptors are located on the surface of each platelet, which take part in the cell-cell and cell-subendothelial matrix interaction. Platelets contain a-granules, which store up to 30 different proteins: ? -thromboglobulin, fibroblast growth factor, factor V, Willebrand factor, fibrinogen, thrombospondin, fibronectin, vitronectin, octomacroglobulin, P-selectin, platelet growth factor (PDGF), tissue plasminogen activator inhibitor type 1, α2-antiplasmin, α1-antitrypsin, protein S, leukocyte chemotactic factor, high-molecular-weight kininogen, etc.. The involvement of α-granule proteins in physiological and pathological processes is multifaceted:


    - mitogenic and chemotactic effects;

    - adhesive action, modulation of platelet aggregation;

    - Involvement in plasma hemostasis;

    - vasoactive effect;

    - immune effect.


 Platelets are fixed on their surfaces and connect with each other due to plasma proteins - adhesion molecules - Willebrand factor, fibronectin, vitronectin, etc.


    The main function of Willebrand factor is to mediate platelet adhesion and subsequent thrombus aggregation.

    Considering the peculiarities of the Willebrand factor, we can say that it performs the function of a "biological glue", fixing platelets on the damaged surface.


    During aggregation, platelets attach to each other and fixate to the damage area. As a result, a thick layer of platelets reinforced by fibrin is formed. This process underlies the formation of the thrombus. Fibrinogen is a unique molecule with the ability to polymerize quickly and form a solid volumetric structure. The formed fibrin matrix is an autologous biocompatible 3D framework, which promotes normal cellular infiltration of monocytes, fibroblasts and other cells playing an important role in wound healing . The advantages of fibrin, compared to other biomaterials, make it ideal for tissue engineering applications. It is a natural nanostructure that enables hemostasis after tissue injury and provides a temporary scaffold that promotes cellular activity as well as the formation of a new extracellular matrix. The morphological and molecular configuration of BOTP has been studied, which is a network of fibrin around platelets that supports the regenerative matrix . The formation of the final clot occurs on the 10th-15th minute after fibrin polymerization.

    Blood clot retraction is its thickening with the release of excess serum from it. Retraction of the clot occurs due to myosin fibrils located in the cytoplasm of platelets. Gradual "shrinkage" of cytoplasm leads to compaction of the whole clot. Mechanisms aimed at limiting clot growth are activated, and gradual dissolution of the clot occurs. This function is carried out due to the system of fibrinolysis.



Autologous BOTP is biocompatible and safe for the patient, as it is obtained from the patient's own blood.


In addition to autologous BOTP there is heterologous BOTP, i.e., plasma obtained not from the patient's blood [130]. At present, OMJ Pharmaceuticals Inc. (USA) has synthesized and produced a heterologous growth factor - bechaplermin, which contains the gene of beta-chain BOTP, manufactured using DNA-technology and embedded in the Saccharomyces mold cell. The indications for its use are the same as those of autologous BOTP, but in spite of this, bechaplermin has significant disadvantages compared to autologous BOTP, namely, the possibility of cross-immune reactions and allergies


A number of experimental works have shown that BOTP, as well as growth factors and other biologically active substances contained in it, accelerate cell proliferation and migration.


Thus, the unique properties of BOTP allow us to consider it as a biocompatible autogenous material representing a fibrin network connecting the wound edges, providing their mechanical support by creating three-dimensional networks, making the processes of tissue settlement, proliferation and differentiation predictable.



















Technologies for obtaining platelet-rich plasma


To date, there have been discussions about the protocols for obtaining BOTP. The design and used laboratory equipment varies greatly, which is due to the lack of standardized technology.


In obtaining BOTP, we can identify common key points: blood sampling, use of anticoagulant, two-step centrifugation using different systems.


Autologous BOTP can be prepared only from whole unconjugated blood, which is taken by venipuncture before the operation. This should be done because platelets are concentrated in the surgical area, initiating coagulation and healing. At the same time, their concentration in the blood decreases slightly.


The concentration of platelets decreases even more if hemodilution occurs during surgery due to the intravenous administration of fluids.


There are several types of anticoagulants, the use of which can achieve platelet release.


Recommended for use are:


- ACD-A (citrate hemoconjugate solution with dextrose);

 - Sodium citrate.

Not recommended for use:

- Heparin (inhibits the release of PRP growth factors);

- EDTA (damages platelet membrane).


Despite the seemingly large selection of methods of obtaining plasma, most of the available commercial systems are expensive and labor-intensive, and only some of them are certified and approved for use in the domestic market.


Centrifugation separates plasma and platelets from erythrocytes and leukocytes. Erythrocytes (7.5 µm), being larger and heavier than platelets (2 µm), settle at the bottom of the test tube as a red sediment. The upper layer, which is yellowish in color, is called platelet poor plasma (PPP). Platelet rich plasma (PRP) containing platelets and leukocytes is formed on the border of the erythrocyte layer and plasma.


After BOTP is obtained, it is recommended to use it within 2-4 hours, although it will be just as effective within 5 days. Experimental studies on the storage mode of BOTP have now been conducted. According to the works published by Eduardo Anitua et al. in 2013 and 2015, it is possible to store the obtained BOTP at +4° - +6° C for 4-5 days, or at -20° C up to 6 months, with preservation of its properties.


Currently, there is an ongoing scientific debate about the protocols for obtaining autologous BOTP. The designs of proposed studies, assessment of clinical and functional results, and the laboratory equipment used vary widely [62], which explains the lack of standardized methods of obtaining and applying BOTP.


The American Association of Blood Banks guidelines indicate that BOTP is obtained from whole blood using low-speed centrifugation, and then platelets are concentrated using high-speed centrifugation, followed by "manual" removal of the supernatant, consisting of blood plasma.


Many protocols provide double centrifugation (in two steps): 50 ml of venous blood is collected, mixed with 6 ml of sodium citrate, and centrifuged at 1500 rpm for 15 minutes.


The resulting BOTP is manually pipetted and mixed with 1/8 volume of sodium citrate, then centrifuged again at 3500 rpm for 10 minutes [97]. According to the authors, regardless of the centrifugation speed and centrifugation time, the separation of erythrocytes and platelets in one step is impossible. And it is during the second stage that the final separation of leukocytes and platelets with a small number of erythrocytes into BOTP and platelet-poor plasma occurs.


In other protocols, 36 ml of blood is drawn and BTP is obtained by single centrifugation (for 6-8 minutes at 1000-2300 rpm), addition of a procoagulant containing 10% CaCl2 solution to activate platelets. 5000 units of prothrombin are added as a clotting factor.


 According to PRP technology blood is drawn in a volume of 18-36 ml using a peripheral venous catheter in 2-4 specialized prp tubes. The number of tubes depends on the area defined for treatment. The tubes are placed in a centrifuge, which is adjusted to the rotation parameters of 3200 revolutions per minute for 5 minutes. The special tube has a fine-dispersed sodium heparin on the inner wall and a separating olefin gel on the bottom. The separating gel separates the red blood cells from the platelets in the plasma. A syringe (2.0- 3.0 ml) takes the supernatant - platelet autoplasma, which is located at the top of the tube above the separating gel.


 It should be noted that low-speed and then high-speed centrifugation complicates the technique and causes mechanical and chemical damage to platelets, and the "manual" processes of obtaining BTP are not clearly regulated (transfer of layers from tube to tube is performed "by eye" using a syringe, pipette), as a result, the accidental obtaining of plasma with an understated platelet count is not excluded. The separating gel present in the tubes adsorbs the cells, resulting in BTP with low platelet content.


Thus, to date, a variety of technically complex and expensive techniques for the preparation of BOTP forces the practitioner to search for the most optimal, simple and affordable procedure using small volumes of blood. Scientific and practical interest is the collection of plasma by one-step closed centrifugation.




Surgical treatment of idiopathic macular rupture with platelet-rich plasma (background)




The first attempts to close IMR surgically using BOTP were made by Gaudric A., et al. in 1995. The study included 40 patients. In 20 patients BOTP was injected at the end of surgery (Group 1). 20 patients were operated according to the standard technique (Group 2). After 11 months of follow-up there were 19 tears closed in the 1st group, but only 13 in the 2nd group [75]. Korobelnik J.F. et al. (1996) included in the study patients with recurrent UTI. After 7 months of follow-up, 87.5% of tears were closed.


Paques M., et al. (1999) conducted a prospective, randomized study on 110 eyes, using BOTP as an adjunct to surgery for IMR in 53 cases. Anatomical closure occurred in 98% with the use of BOTP and in 82% without it. However, the authors believed that obtaining BOTP requires certain additional efforts, increases the cost of intervention and, most importantly, in a randomized multicenter study, no significant improvement of functional outcomes of treatment was found.


Velhagen K.H. et al. (1999) tried to find out the mechanism of action of various blood components on pigment epithelium cells. They conducted the first experimental study in which they demonstrated the influence of BOTP, blood serum and PDGF (plateletderived growth factor) on cell migration and proliferation [154, 155]. Pigment epithelium was cultured in DMEM (Dulbecco's Modified Eagle Medium - medium used for cultivation of a wide range of animal and human cells). As a result, an increased cell proliferation was noted in the group using BOTP at all stages of incubation period (from 1 to 5 days), suggesting that it induces proliferation and migration of pigment epithelium cells.


 A number of experimental works have shown that BOTP, as well as growth factors and other biologically active substances contained in it, accelerate glial cell proliferation and migration.



Thus, Castelnovo L. et al. (2000) conducted an experiment in vitro and demonstrated the proliferative and chemotactic effect of BOTP on glial cell structures. Studying the effect of growth factors, the authors concluded that only fibroblast growth factor (FGF) causes strong glial and mitogenic responses (P<0.001). The biological activity of FGF is diverse. Being a mitogen for various cells of neuroectodermal and mesenchymal origin, it supports and stimulates cell differentiation of various neuronal types in vivo and in vitro. According to the authors, surgical treatment of IMR based on the combination of vitrectomy with the use of blood BOTP is a successful and effective way of treatment.



 Recent studies of glial tissue suggested the participation of Müller cells in the restoration of damaged retina by IMR closure.


It should also be noted the foreign studies aimed at studying the regeneration of retinal glial tissue, in particular Müller cells, which demonstrate that they have an evolutionary potential and can serve as a basis for the lost nerve fibers.


Mulhern M.G. et al. (2000) described a study to determine whether treatment of IMR with vitrectomy and administration of BOTP and short-term tamponade with SF6 gas would be as effective as long-term tamponade with C3F8 gas. Group 1 patients underwent vitrectomy, administration of BOTP and C3F8 gas, and were recommended to be face down for 2 to 4 weeks. Group 2 patients were operated similarly, except that SF6 gas was used and they were face down for 1 week. After 3 months in the postoperative period, the average improvement in visual acuity was similar in both groups. Anatomical success in group 1 was 96.7% and in group 2 was 93.5%.


Garcia-Arumi J.1. et al. (2001) proposed to remove the VPM before BotP application in patients with high degree myopia, obtaining MR closure in 86% of eyes.


Comparative studies continued to be conducted to find the most effective blood components used in IMR surgery. In 2001 Hoerauf H. et al. once again proved the advantage of BOTP . The authors reported a 94% anatomical success rate after BOTP injection, compared to 36% success rate in the group after autologous whole blood injection. Removal of VPM was not performed believing that it minimizes retinal trauma.


Kube T. et al. (2002) showed improvement of functional results after removal of an ERM, which was preceded by indocyanine green staining (ICG), obtaining closure of the tear in 94% of cases.


However, not all surgeons recognized the benefit of removing the VMM. Thus, Cheung C.M.G. et al. (2005) in the experience of 56 patients showed that after vitrectomy with application of BOTP without removing the TMF it was possible to achieve closure of the tears in 98% of cases (55 patients). According to the authors, the removal of the VPM increases the risk of hemorrhages, retinal tears, and also possible formation of ERM in the postoperative period.


According to Kapoor K.G. et al. (2012), the use of BOTP and removal of VPM complement each other and provide a good result without the patient's face-down position and gas bubble pressure on the tear edges. Thirteen patients were included in the study who could not comply with the forced face-down position for medical reasons.


 The duration of some UTIs was more than 2 years. The surgery was performed according to the standard technique with mandatory removal of the TMJ, administration of BOTP and C3F8 tamponade. As a result, 100% of the ruptures were closed.


Konstantinidis A. et al. (2013) concluded that the size of the BMI and the presence of the VMM does not affect the surgical outcome, and the use of BOTP and a strict face-down position are crucial factors in achieving the anatomical effect. The authors operated on 21 patients, performing vitrectomy without removing the VMM, obtaining tear closure in all cases.


Engelmann K. et al. (2015) conducted a study devoted not only to the issue of surgical treatment improvement. Clinical observations showed that the volume of BOTP has no effect on the outcome of surgery, and its effect is to provide high content of growth and trophic factors, which have a pronounced stimulating effect on the processes of tissue repair. In vitro study showed that both BOTP and PDGF significantly increase the movement and proliferation of pigment epithelium cells.


In Purtskhvanidze K. (2018), where BOTP was used during reoperations, in a retrospective analysis of OCT - studies, there was a relatively large ellipsoidal defect that significantly exceeded the preoperative minimal diameter of the IMR (550 and 434 µ m on average, respectively).


Blood serum have been attempted in IMR surgery. However, the most significant positive effect was given by BOTP, which was associated primarily with a high content of growth and trophic factors having a pronounced stimulating effect on tissue repair processes.


It was also suggested that fibrinogen contained in the plasma, when transformed into fibrin, provides mechanical support for the tear edges by creating three-dimensional networks serving as a basis for the processes of cell proliferation and differentiation in the fovea zone.


Thus, in 1995-2005, autologous blood BOTP was successfully used in the surgical treatment of IMRs.


The existing foreign studies demonstrate its safety and efficacy.


In the first years of BotP use, anatomical closure of the rupture was achieved in 94-98% of cases. In the next few years, further improvement of anatomical and functional results of IMR treatment was provided by the improvement of instrumentation and introduction of new technologies, such as VMM removal; there were almost no new reports on the use of BOTP. The designs of the studies performed, patient groups, and assessment of clinical efficacy varied greatly. In addition, the sample size was small and limited the generalization of results. The analysis was conducted retrospectively, describing only statistical data on the number of positive and negative cases. The conducted studies were of a factual nature without an attempt to assess the effect of blood BOTP on retinal microstructural changes occurring in the macular area after surgical treatment. To date, structural changes in the macular area when using BOTP have remained virtually unstudied in the literature. Only in one study where BOTP was used during reoperations, a relatively large ellipsoidal defect significantly exceeding the preoperative minimum diameter was observed in retrospective analysis.





















PRP Platelet Rich Plasma


What is PRP? How does PRP therapy work? What is the success of PRP treatment in Munich? Where does PRP help?



What is PRP?


PRP stands for "Platelet Rich Plasma". Our blood contains a multitude of living cells. One type of cell are platelets or thrombocytes. PRP is concentrated blood plasma from a single patient (i.e. blood without red blood cells). high number of platelets, growth factors and proteins The white blood cells also contained play a somewhat less important role in PRP (for more information on PRP components and PRP production see below). for rejuvenation and cell regeneration of various body tissues depending on the type of application with the addition of medication, etc. Cells activated in PRP are processed, injected or massaged into the skin, connective tissue, scars, muscles, joints and roots and provide regeneration and revitalization, a so-called autologous Cell regeneration (for those particularly interested: a lot of information on the scientific background of PRP and the vampire lift at the end of this article. Many ideas and techniques of PRP development come from research with stem cells.


Application of PRP


PRP is used in many areas today: to beautify and regenerate the skin, as a lifting method, to promote hair growth, for wound healing, in orthopedics and sports medicine ... As a dermatologist, I prefer to treat the face, neck, décolleté and scalp But we can use it to regenerate almost any aged tissue


- Face: intradermal PRP for wrinkle injections, PRP therapy for volume enhancement

- Neck: PRP for wrinkle treatment back of the hand: PRP for rejuvenation.

- Décolleté: plasma and PRP injections to tighten the skin, reduce wrinkles (similar to vampire lift).

- Scalp: PRP for hair growth, regeneration, hair thickness growth, intradermal. PRP injection (see there for more information on PRP and hair loss).

- Stretch marks, stretch marks: PRP for skin tightening and regeneration.(possibly in combination with laser, puncture) to improve stretch marks

- Operation scars: PRP to improve scars, wound healing

- Acne scars: PRP to soften scars, PRP to tighten skin (possibly in combination with laser, puncture) to soften skin.


PRP and fresh cell therapy


Fresh cell therapy was a forerunner of PRP. Until the 1980s, unsuitable methods were used to attempt to use animal growth factors and stem cells for cancer therapy and regeneration. At that time, numerous celebrities such as Pope Pius XII, the Japanese, Saudi and Ethiopian emperors, Charlie Chaplin, Federal Chancellor Konrad Adenauer and the national soccer coach Helmut Schön treated themselves. Today, of course, we no longer work with the problem of fresh sheep cells, but with the patient's own cells so that the dangers no longer exist. 


PRP und Doping


PRP has been used for regeneration in sports medicine and orthopedics for years. It is estimated that around half of all football and basketball clubs in the German Bundesliga use PRP to shorten the recovery time of their stars after injuries. Ralph Nadal, Kobe Bryant, the German basketball team and others, FC Barcelona doctor Dr. According to his own Twitter posts in 2014, Cugat treated the injured Bayern star Thiago with PRP. 2011 and therefore still controversial in sports medicine. Reason: doping researchers cannot differentiate between natural PRP and artificial growth factors. In sports medicine, the effect of increasing the performance and regenerating PRP and autologous blood is not very controversial.


Risks and side effects of PRP


The risks of the treatment are very low, since the patient is treated with the patient's own blood. Small bruises, redness, and swelling are rarely possible. The puncture of the injection needle is of course noticeable, but hardly hurts. Contraindications: When is PRP not possible? Blood borne diseases are contraindications to PRP; To be on the safe side, we don't do PRP for hepatitis, HIV and others. Pregnant women should not be treated. You have to be very careful with neurodermatitis or psoriasis on the face, in general with all acute or infected skin diseases. Blood clotting disorders, autoimmune diseases, liver diseases and cancer are also more likely to speak against it. At least three days before treatment with PRP (better 710 days) you should not take aspirin, ASA, Volaren, ibuprofen or similar pain. and anti-rheumatoid drugs (NSAIDs), as they reduce the vitality of blood platelets. Paracetamol and Novalgin / Metamizol are allowed (by the way, this information is good evidence from the experience of the treating doctor: if he does not tell you, you should have the treatment carried out by someone else (the doctor does not seem to have really investigated what it is).


Implementation PRP


To make PRP, a small amount of blood is taken from the vein and processed using a special process; The doctor separates the red blood cells from the blood plasma; the effective part for the PRP is taken; then the PRP can be injected. in pure form or mixed with medication, or even hyaluronic acid with the best of needles. PRP can also be massaged through the fine channels of the skin after a laser or microneedle treatment. Treatment takes approximately 45 minutes, including blood collection, preparation, and treatment. PRP, its growth factors and stem cells strongly stimulate cell renewal, wound healing and the regeneration of old used cells. Smooth skin and wrinkles are rejuvenated by PRP. The elasticity, color and firmness of the skin are improved by a rejuvenating treatment with the body's own blood PRP factors.For wrinkle treatment and vampire lifting, PRP can be combined with hyaluronic acid and other firming substances, the filling effect is stronger, the tolerance better and the effectiveness longer. Immediately after the treatment you will notice slight swelling and hardening of the skin. In the days and weeks that follow, the growth factors of the PRP develop their biological effect to strengthen the skin and the surrounding tissue. The success of any PRP therapy increases with the number of applications, is stronger after a few weeks and lasts for up to 18 months. The annual renewal with PRP stabilizes and strengthens the success.


Combination PRP with laser, microneedling, surgeries


There are several ways to inject cells and PRP growth factors deep into the skin. During the vampire facelift, they are injected with the finest needles. Another way to tighten the skin is to apply PRP without a needle. But only PRP. Dig deeper if small micro-openings have been opened in the upper layers of the skin in a personal consultation, which combination of PRP can best solve your problem. PRP can also be used on previously operated skin and enhance the effect of a face lift. 


PRP variants Internet


The term PRP is not legally protected. The tricyclopathic plasma production process can be more or less laborious; the number of living platelet cells and growth factors in PRP varies accordingly, as does the effect of PRP. I know 15 different methods of making PRP that are constantly evolving. Of course, we choose the most effective PRP method for our method. Depending on the technology used, PRP contains between 40% and 95% concentrated platelets; according to the FDA (American health authority), a concentration of 50. Speaking of PRP However, contrary to the claims of many manufacturers, the effectiveness is not necessarily linked to the concentration, because highly concentrated PRP is not more effective than slightly less concentrated PRP.


More important is the total number of platelets. Platelet concentrations approximately two to three times normal plasma concentrations appear to be the optimal level. Of course, the type and amount of additives as well as the technical process also influence success.Since approx. 95% of the effective growth factors are secreted by living platelets in a narrow time window, the application after the PRP extraction must take place in precisely this time window. On the Internet you can find treatment prices between 300 euros and 1000 euros. for PRP. In our opinion, one should be careful with cheap offers, as the production is often not done correctly. Even "PRP from plant extracts" is scientifically advertised under the term PRP, of course complete nonsense, since plants do not contain platelets in the blood. Some well-known doctors charge too high prices for PRP therapy. I wonder if this will make it more effective.


Cost PRP


A treatment with PRP costs between 500, 800 euros and depends on the amount of PRP obtained, the duration of the treatment, the additives used, etc. It is sufficient to maintain and stabilize the success. Wrinkle prevention with PRP is also very effective; From the mid-twenties, a one-time annual treatment is sufficient. Women and men over 45 years of age often combine the treatment with other treatments, for example with microneedles. The rejuvenation and stretching effect is then faster and more visible. Wrinkle prevention is also very effective; A one-time annual treatment from the mid-twenties is sufficient. Many skin damages, such as the consequences of sunburn, eczema, dry skin, can be treated preventively with PRP.


Scientific background of PRP


Many aging processes and diseases occur due to the body's inability to replace dead or diseased cells or due to insufficient speed. the face loses volume, sags and looks haggard. Scientists have long tried to cure these processes using the body's own growth factors and the so-called stem cells; These are "mother" cells that can become almost any other "adult" cell. The embryo is made up of only these stem cells.


These wonderful healing cells are also found in the body's own fats and serums. Platelets in the blood can have a similar effect. They have this effect on wound healing and bleeding in the body, but are present in the blood only in small amounts, which can be obtained in concentrated form from the blood in PRP. The following growth factors, among others, can be found in PRP: TGFbeta: promotes cell growth, blood vessels, extracellular matrix formation.


PDGFAB: stimulates cell growth, regulates fibroblasts. BFGF: stimulates fibroblasts. IGF: promotes collagen synthesis, cell proliferation, fibroblast migration. ) required efficacy or viability testing ((pselectin expression after ADP-specific stimulus; against hypotonic stress; supplementation in response to collagen) prior to approval of the PRP kit.After injection, vital growth factors are immediately active., platelet concentrate continues to produce new growth factors still within 7 days, after death, they are replaced by other growth factors, absorbent cells (macrophages), which perform similar functions.


For example, when platelets are activated by a wound, their release leads to direct and indirect tissue regeneration and wound healing; they also recruit other cells such as fibroblasts, mesenchymal stem cells and leukocytes; They also serve for healing and regeneration. New collagen, elastin, new blood vessels and young cells are formed.



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