Advantages and Clinical Applications of Lyophilized Platelet-Rich Plasma in Various Disease Conditions: A Review

Platelet rich plasma (PRP) is a biological product defined as a portion of the plasma fraction of autologous blood with a platelet concentration above the baseline. The plasma occupies 55% of blood, which is rich in immunoglobulins and proteins that have a wide range of applications in various medical fields. Plasma therapy is applied to tackle various disorders or diseases as it induces the body to develop new healthy cells. It contains important components like antibodies, coagulation factor, enzymes, fibrinogen, proteins and albumin. PRP is a unique and advanced treatment which helps to increases the body’s natural healing process. Platelet lysate which is obtained from platelet rich plasma consist of various growth factors such as chemokines, cytokines, and antibacterial molecules and also has anti-inflammatory, immunomodulatory, anti-fibrotic and repairing effects. As PRP is rich in the proteins and several antibodies, it is used for various chronic therapies such as hemophilia and autoimmune disorders as well as in various severe health problems. Lyophilized Platelet-rich plasma (LPRP) therapy is currently used in various fields such as in tissue regeneration, wound healing, scar revision, skin rejuvenating effects, alopecia and for the coronavirus disease (COVID-19). It is also used to heal wounds and illnesses. LPRP therapy is gaining attraction by many health professionals as it is a safe, effective, efficient, and easy approach in procuring, preserving, and therapy. In this review we described the advantages and applications of using lyophilized PRP in various diseases which might found to be effective in different treatment. Keywords: Plasma, Platelet, Growth Factors, Lyophilized platelet rich plasma.

Method to obtain PRP and L-PRP for the use in routine medical practice of orthopedic traumatologist

The paper presents the results of experimental study of two methods for blood processing to obtain platelet-rich plasma (PRP). Due to the widespread clinical use of platelet-rich plasma in orthopedics and traumatology, a differential approach to the use of PRP and leukocyte- and platelet-rich plasma (L-PRP) has become relevant. Most commercial kits for PRP preparation significantly increase the cost of treatment. In addition, the main attention is focused on the concentration of platelets in the final product and the marketing features of a particular kit (separating gel, tube shape, anticoagulant, activator, etc.). The role of leukocytes in the features of the regenerative effect of platelet-rich plasma stays out of focus. The blood of volunteers was processed in two different ways and the cellular composition of the obtained products was analyzed. Sample No. 1 was used as a control for the baseline blood cell composition. Sample No. 2 was centrifuged in a sterile plastic tube, platelet rich plasma was collected manually. Sample No. 3 was centrifuged in a sterile modified syringe, platelet rich plasma was collected using a closed technique by means of connectors together with a rich layer. The results of statistical analysis showed that there were obtained totally different biological products with different concentrations of platelets and leukocytes. The first sample (control) had platelet concentration of (228.69 ± 39.15) × 109/l and leukocyte concentration of (5.18 ± 1.32) × 109/l. In the second sample (tube + manual sampling), platelet concentration was (429.38 ± 79.92) × 109/l and leukocyte concentration — (0.85 ± 0.34) × 109/l. The third sample (syringe plunger + closed vacuum collection) had platelet concentration of (541.15 ± 85.49) × 109/l and leukocytes of (6.56 ± 1.92) × 109/l. The data are given without regard to –12.5% deviation on dilution with citrate dextrose. As the result of the work, orthopedic traumatologists can use the simplest non-commercial methods for obtaining PRP and L-PRP in their practice depending on nosology and clinical tasks.

Case Report: Take a Second Look: Covid-19 Vaccination-Related Cerebral Venous Thrombosis and Thrombotic Thrombocytopenia Syndrome

We present two cases of ChAdOx1 nCov-19 (AstraZeneca)-associated thrombotic thrombocytopenia syndrome (TTS) and cerebral venous sinus thrombosis (CVST). At the time of emergency room presentation due to persistent headache, blood serum levels revealed reduced platelet counts. Yet, 1 or 4 days after the onset of the symptom, the first MR-angiography provided no evidence of CVST. Follow-up imaging, performed upon headache refractory to nonsteroidal pain medication verified CVST 2–10 days after initial negative MRI. Both the patients received combined treatment with intravenous immunoglobulins and parenteral anticoagulation leading to an increase of platelet concentration in both the individuals and resolution of the occluded cerebral sinus in one patient.

Platelets are dispensable for the ability of CD8+ T cells to accumulate, patrol, kill and reside in the liver

Effector and memory CD8+ T cells accumulate in large numbers in the liver where they play key roles in the control of liver pathogens including Plasmodium. It has also been proposed that liver may act as the main place for elimination of effector CD8+ T cells at the resolution of immune responses. Platelets and the integrin LFA-1 have been proposed to be critical for the accumulation of protective CD8+ T cells in the liver; conversely, asialo-glycoprotein (ASGP) expression on the surface of CD8+ T cells has been proposed to assist in elimination of effector T cells in the liver. Here we investigated the contributions of these interactions in the accumulation of CD8+ T cells activated in vitro or in vivo by immunization with Plasmodium parasites. Using Mpl-/- mice with constitutive thrombocytopaenia and antibody-mediated platelet depletion models we found that severe reduction in platelet concentration in circulation did not strongly influence the accumulation and protective function of CD8+ T cells in the liver in these models. Surprisingly, inhibition of ASGP receptors did not inhibit the accumulation of effector cells in the liver, but instead prevented these cells from accumulating in the spleen. We further found that enforced expression of ASGP on effector CD8+ T cells using St3GalI knockout cells lead to their loss from the spleen. These data suggest that platelets play a marginal role in CD8+ T cell function in the liver. Furthermore, ASGP-expressing effector CD8+ T cells are retained in the liver but are lost from the spleen.

Cell Attachment Capacity and Compounds of Fibrin Membranes Isolated from Fresh Frozen Plasma and Cryoprecipitate

Fibrin membranes are widely used in regenerative medicine because they are biocompatible, biodegradable, contain growth factors, and support cell attachment. Most commonly they are produced from serum, but they can also be isolated from activated plasma. To increase the fibrinogen concentration of plasma, cryoprecipitate isolation is a possible solution. In this work, cryoprecipitate was prepared from fresh frozen plasma, isolated by plasmapheresis. The concentration of cellular elements, fibrinogen, total protein, and immunoglobulins among others was measured in different concentrations of cryoprecipitates. After activation with Ca-gluconate, fibrin membranes were produced in different thicknesses, and human mesenchymal stem cells were seeded onto the membranes. They were visualized by live-dead staining and their viability was determined by XTT. The platelet-derived growth factor AB content was quantified by ELISA. Our results showed that fibrinogen and platelet concentration can be multiplied in plasma by cryoprecipitate isolation, which affects the thickness and slightly the growth factor content of the membranes. According to live-dead staining, the thickness of the membranes does not influence cell attachment, and XTT measurement did not reveal a significant difference in cell attachment capacity either; however, a growing trend could be observed in the case of some membranes.

Simple Tube Centrifugation Method for Platelet-Rich Plasma (PRP) Preparation in Catalonian Donkeys as a Treatment of Endometritis-Endometrosis

The aim of this study was to standardize a simple, manual platelet-rich plasma (PRP) protocol in Catalonian donkeys using single-spin tube centrifugation as a treatment for jenny endometritis. The objective was to obtain a blood product with a moderate concentration of platelets (2 or 3 times baseline physiologic values) and a low WBC (White Blood Cells) concentration. Blood was drawn from six Catalonian donkeys using acid citrate dextrose (ACD) as an anticoagulant, and then processed by single centrifugation at 133× g for two different centrifugation times (10 and 15 min). The PRP samples were evaluated by flow cytometry, and TGF-β1 (Transforming Growth Factor-Beta1) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). The 10 min centrifugation protocol resulted in a slightly greater release of TGF-β1 (6044.79 ng/mL), a 2.06-fold increase in platelet concentration, and a 15-fold reduction in leukocyte concentration when compared to the initial values. The 15 min centrifugation time resulted in a 2.44-fold increase in baseline platelet concentration, a reduction in WBC count by a factor of 20, and slightly lower TGF levels (5206 ng/mL). We conclude that both protocols are adequate for the obtention of PRP, and both may have an acceptable therapeutic potential for use in this species, although this needs to be further validated.

Time-Dependent Cytokine-Release of Platelet-Rich Plasma in 3-chamber Co-culture Device and Conventional Culture Well

Platelet-rich plasma (PRP) contains bioactive cytokines to enhance tissue healing. The best PRP preparation protocol and timing of the treatment have not been determined yet. To screen the best-fit PRP, a 3-chamber co-culture device was developed. We hypothesized the concentrations of the cytokines from different PRPs in the co-culture plates had a high correlation with those in conventional 24-well culture plates at different time points. The concentrations of the cytokine from PRPs would be correlated with platelet concentrations. The correlation of transforming growth factor beta-1 (TGF-β1) and platelet-derived growth factor AB (PDGF-AB) in both devices were compared at 0, 24, 48, 72, and 96 h from two PRPs as well as that of platelet and cytokines concentrations. The results revealed that there was a moderate to high correlation in TGF-β1 concentrations between the 3-chamber co-culture and conventional culture device until 96 h. The correlation of PDGF-AB concentrations in both devices had moderate to high correlation in the first 24 h, and then it became modestly correlated from 48 to 96 h. A high correlation was found between platelet and TGF-β1 concentration at 96 h. However, they were modestly correlated in other time points. A negative or modest correlation was found between platelet and PDGF-AB concentration in all time points. In conclusion, TGF-β1 and PDGF-AB revealed a time-dependent manner of release at five time points. There is a moderate to high correlation of the TGF-β1 and PDGF-AB concentration in both devices at different time points. However, TGF-β1 and PDGF-AB concentrations are not always proportional to the platelet concentration of the PRPs.

A cross-sectional analysis of the effects of various centrifugation speeds and inclusion of the buffy coat in platelet-rich plasma preparation

Introduction: Platelet-rich plasma is an autologous blood preparation which is used in various medical specialties because of its regenerative properties. There is a wide variation in platelet-rich plasma preparation protocols and attaining the ideal platelet yield (>1 million platelets/μL) in a clinic setting can be challenging. We aimed at analyzing the centrifuge spin rates at which to attain an ideal platelet-rich plasma yield and also to study the effect of inclusion of the buffy coat after the first spin on the final platelet concentration in platelet-rich plasma. Methods: Seventy-five whole blood samples were obtained and divided into two groups – (1) leukocyte-rich platelet-rich plasma group and (2) leukocyte-poor platelet-rich plasma group. Samples in both groups were centrifuged using the dual spin method, at one of three centrifugation speed combinations (initial “soft” spin and second “hard” spin speeds, respectively): (1) 100 g/400 g, (2) 350 g/1350 g and (3) 900 g/1800 g. Platelet, red blood cell (RBC) and white blood cell (WBC) counts in both groups were compared. Results: The 100 g/400 g spin gave a high platelet yield (increase of 395.4 ± 111.1%) in the leukocyte-poor-platelet-rich plasma group, while in the leukocyte-rich platelet-rich plasma group both 100 g/400 g and 350 g/1350 g spins resulted in significantly higher yields with an increase of 691.5 ± 316.3% and 738.6 ± 193.3%, respectively. Limitations: The study was limited by a smaller sample size in the pure platelet-rich plasma (leukocyte-poor platelet-rich plasma) group. Conclusion: Ideal platelet yields can be achieved with both the 100 g/400 g as well as the 350 g/1350 g spins using the buffy coat inclusion method while the 100 g/400 g spin for “pure” platelet-rich plasma accomplishes a near-ideal platelet count with significantly reduced contamination with other cells.

Efficacy of i-PRF in regenerative endodontics therapy for mature permanent teeth with pulp necrosis: study protocol for a multicentre randomised controlled trial

Background Dental pulp necrosis, a common health problem, is traditionally treated with root canal therapy; however, it fails in restoring the vitality of damaged pulp. Most studies regarding regenerative endodontic therapy (RET) are limited to the treatment of immature necrotic teeth. Given that injectable platelet-rich fibrin (i-PRF) has shown great potential in regenerative medicine as a novel platelet concentration, this study is designed to explore whether i-PRF can serve as a biological scaffold, extending the indications for RET and improving the clinical feasibility of RET in mature permanent teeth with pulp necrosis. Methods This is a randomised, double-blind, controlled, multicentre clinical trial designed to evaluate the clinical feasibility of RET for mature permanent teeth with pulp necrosis and to compare the efficacy of i-PRF and blood clots as scaffolds in RET. A total of 346 patients will be recruited from three centres and randomised at an allocation ratio of 1:1 to receive RET with either a blood clot or i-PRF. The changes in subjective symptoms, clinical examinations, and imaging examinations will be tracked longitudinally for a period of 24 months. The primary outcome is the success rate of RET after 24 months. The secondary outcome is the change in pulp vitality measured via thermal and electric pulp tests. In addition, the incidence of adverse events such as discolouration, reinfection, and root resorption will be recorded for a safety evaluation. Discussion This study will evaluate the clinical feasibility of RET in mature permanent teeth with pulp necrosis, providing information regarding the efficacy, benefits, and safety of RET with i-PRF. These results may contribute to changes in the treatment of pulp necrosis in mature permanent teeth and reveal the potential of i-PRF as a novel biological scaffold for RET. Trial registration ClinicalTrials.govNCT04313010. Registered on 19 March 2020

Are Tryptophan and Serotonin Alteration Act as a Trigger Biomarker for Type 2 Diabetes Complications?

Tryptophan (Trp) is a vital amino acid causing clinical implications in the human body and biological specimens. Serotonin (5-HT) is well-recognized for its biological activities in the brain, and as a familiar neurotransmitter, it regulates various neuropsychological processes. Alterations in the serotonergic system were proved to play a role in the pathogenesis of different neurological and psychiatric diseases. In recent years, Trp and 5-HT levels have been considered as triggers of diabetes mellitus (DM). The altered Trp metabolism may also play roles in the pathogenesis of DM and developing a risk of complications. The whole blood (WB) 5-HT level was mainly lower among diabetic patients compared to others. That is mostly derived from a lower platelet concentration of 5-HT in these patients. Indeed, 5-HT level can be considered as a potent biomarker for early detection of DM complications. Besides, it was proved that outside the digestive and central nervous systems, 5-HT was discovered in beta cells, and scientists have been attempting to realize its mechanism of action ever since. Towards to end, the determination methods, biomarker’s role, and approaches of 5-HT and Trp levels were thoroughly investigated in both healthy and diabetic patients with or without complications. Moreover, the association between insulin and 5-HT has been specifically discussed. Our study concluded that Trp and 5-HT levels could be exclusively applied for early diagnosis of DM complications as well as many other complications.