Platelet Count In Platelet-rich Plasma Research Articles

Integration of platelet features in blood and platelet rich plasma for detection of lung cancer

ObjectivesTo determine whether the integration platelet features in blood and platelet rich plasma can establish a model to diagnose lung cancer and colon cancer, even differentiate lung malignancy from lung benign diseases. Methods245 individuals including 159 lung cancer and 86 normal participants were divided into the training cohort and testing cohort randomly. Then, 32 colon cancers, 37 lung cancers, and 21 benign patients were enrolled into validate cohort. The whole blood and corresponding platelet rich plasma (PRP) samples from all participants were prospectively collected, and the platelet features were determined. The features which are statistically significant at the univariate analysis in the training cohort and reported significant features were entered the diagnostic model. A receiver operator characteristic (ROC) curve was drawn to evaluate the accuracy of the model in each cohort. ResultsIn the training cohort, multiple platelet features were significantly different in lung cancer patients, including MPV in whole blood, MPV, and platelet count in PRP and platelet recovery rate (PRR). For the training cohort, the diagnostic model for lung cancer performed well (AUC = 0.92). The probability distribution of lung cancers and controls in testing cohort were also separated well by the diagnostic model (AUC = 0.79). The diagnostic model for colon cancer also performed well (AUC = 0.79). The model also has a potential value in differentiating the lung malignancy from the benign (AUC = 0.69). ConclusionThe PRR was first raised and used in the detection of lung cancer. This study identified a diagnostic model based on PRR and other platelet features in whole blood and PRP samples with the potential to distinguish patients with lung cancer or colon cancer from healthy controls. The model could also be used to distinguish between lung cancer from the benign disease.

High Intensity Interval Exercise Increases Platelet and Transforming Growth Factor‐β Yield in Platelet‐Rich Plasma

Platelet-rich plasma (PRP) is an emerging orthobiologic treatment for musculoskeletal conditions like osteoarthritis. Two studies have demonstrated the influence of longer duration exercise on PRP composition, but no study has ever explored the impact of high intensity interval exercise (HIIE) on PRP content. To quantify cellular and growth factor content changes in PRP after 4 minutes of HIIE. Controlled laboratory pilot study. Academic sports medicine center. Ten healthy volunteers (5 male, 5 female). Volunteers had PRP prepared from 15 mL of whole blood using a single spin, plasma-based system (autologous conditioned plasma [ACP]) immediately before and after 4 minutes of HIIE on a stationary exercise bike (Tabata protocol). The PRP was sent for complete blood counts and enzyme-linked immunosorbent assay (ELISA) to quantify transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)-1, and vascular endothelial growth factor (VEGF). Mean platelet count in PRP increased from 367.4 ± 57.5 k/μL to 497.7 ± 93.3 k/μL after 4 minutes of HIIE (P < .001). TGF-β also increased from 8237.2 ± 7676.5 pg/mL to 21 535.7 ± 4062.6 pg/mL postexercise (P = .004). The other cellular components (leukocytes, red blood cells, and mean platelet volume) and growth factors (PDGF, IGF-1, and VEGF) were not significantly changed. A short 4-minute bout of HIIE significantly increased the total platelet count and TGF-β concentration in PRP.

Platelet-rich plasma injection for the treatment of atrophic fracture nonunion

To explore clinical effects of platelet rich plasma (PRP) injection in treating atrophic fracture nonunion. From March 2015 to March 2017, 15 patients with atrophic fracture nonunion were treated with PRP injection, including 10 males and 5 females, aged from 23 to 56 years old with an average age of (40.0±9.1) years old, the time of fracture nonunion ranged from 6 to 14 months with an average of (8.87±2.45) months. Preparing PRP by extracting 60 to 100 ml peripheral blood. PRP platelet count ranged from 587 to 1 246 with an average of (947.13±158.58) ×10 9 /L. Under the perspective, 13 to 20 ml PRP were injected into the fracture end, and each injection was performed once on the first and the second week of the treatment. Complications such as whether the limb was shortened, angulation, and rotational deformity and radiological examination were observed. All patients were followed up from 6 to 12 months with an average of (6.8± 2.1) months. No shortening, angulation, and rotational deformity occurred. Thirteen patients had fracture healing, the time ranged from 4 to 6 months with an average of (4.8±0.7) months. Two patients had no completely porosis at 12 months during following up, and 1 patient occurred bolt loose. Other patients had no complications. The stability of fracture ends of atrophic fracture nonunion after internal fixation is an indication for local PRP injection. PRP treatment for atrophic fractures could completed under local anesthesia, and it has advantages of safe operation and reliable efficacy.

Comparison of platelet counts in platelet-rich plasma (PRP) obtained by two methods from dogs

Platelet-rich plasma (PRP) is widely used in human medicine, but seldom used in veterinary practice. Even so, the efficacy of PRP in osteoarthritis, dental implants, bone defects and wound healing in dogs and horses is well documented. There are many commercially available PRP tubes and kits for horses and humans, but studies on the effectiveness of these products in dogs are limited. Researches have demonstrated that systems developed for humans and horses do not always produce the same results in dogs. In the present study, two prevailing systems, a sodium citrate-based system and a gel-based system, were compared in terms of their platelet output. The data obtained revealed that, although there was no statistically significant difference between the two systems, the sodium citrate-based kit is probably more efficient in practice due to its superiority in volume output. PRP is an important component of regenerative therapy. It is autologous and practical, but has not been sufficiently implemented in veterinary practice, but promising clinical satisfaction and must be considered in various conditions.

Assessment of Quality of Platelet-Rich Plasma Produced With Desktop Centrifuge and Comparison With Standardized Commercially Available Platelet-rich plasma.

ABSTRACTAims:The aim of this study was to assess the quality of platelet-rich plasma (PRP) produced by in-house desktop centrifuge method and compare it with that of standardized commercial PRP.Materials and Methods:REMI desktop centrifuge was used to prepare PRP and to compare with standardized commercial PRP by calculating the quantity of platelets using Beckman Coulter cell counter in 10 PRP samples and assessing the morphological quality of platelets using JEOL JEM transmission electron microscope (TEM).Statistical Analysis Used:The t test for platelet count in desktop PRP with the test value of therapeutic PRP was 12.618. The P value was <0.001, which was significant statistically. The data followed a normal distribution in normal Q-Q plot for platelet count in desktop centrifuge. So the test samples were not much deviated.Results:The platelet count was lesser than that of standardized commercial PRP. When viewed under JEOL JEM transmission electron microscope, the α granules in platelets were intact and the morphological quality of the PRP was good.Conclusions:With this study, we have determined that the morphological quality of PRP produced by the in-house desktop centrifuge method is comparable to that of standardized commercial PRP. Though the quantity of platelets was less than 1 million cells/μL, the clinical results were good with desired bone formation, thereby providing good avenue for further research.

Platelet-rich plasma injection for adults with acute Achilles tendon rupture: the PATH-2 RCT

BackgroundAchilles tendon rupture (ATR) has a long healing period, which is challenging for patients and clinicians. Platelet-rich plasma (PRP) is an autologous concentration of platelets thought to improve tendon function recovery. Although preliminary research has indicated positive effects, there is, as yet, no evidence of clinical efficacy from adequately powered robust clinical trials.ObjectivesThe objectives were to determine the clinical efficacy of PRP in patients with acute ATR using an objective mechanical muscle–tendon function measure and patient-reported outcome measures (PROMs), and to determine which PRP components contribute to its mechanism.DesignThis was a multicentre, parallel-group, participant- and outcome assessor-blinded randomised controlled trial (RCT) comparing PRP with placebo. Two embedded substudies investigated the PRP’s quality and composition and its effects on healing tendon tissues.SettingThis trial was set in trauma and orthopaedic surgery departments in 19 NHS hospitals in England and Wales.ParticipantsAdults with acute ATR presenting within 12 days of injury to be treated non-surgically were eligible. Patients with platelet dysfunction or leg functional deficiency were excluded.InterventionsParticipants were randomised 1 : 1 to the PRP injection group or the placebo group (dry needle in the rupture gap) by central computer-based randomisation using minimisation, stratified by centre and age.Main outcome measuresThe primary outcome measure was the Limb Symmetry Index (LSI) of work during the heel-rise endurance test at 24 weeks. Secondary outcomes measures, collected at 4, 7, 13 and 24 weeks, were repetitions, maximum heel-rise height, Achilles tendon Total Rupture Score (ATRS), quality of life (as measured using the Short Form questionnaire-12 items version 2), pain and participant goal attainment. Needle biopsies of the affected tendon zone were taken under ultrasound guidance at 6 weeks from 16 participants from one centre. Whole blood was analysed for cell count. PRP was analysed for cell count, platelet activation and growth factor concentration. The primary analysis was intention to treat.ResultsA total of 230 participants were randomised: 114 to the PRP group (103 treated) and 116 to the placebo group (all treated). One participant withdrew after randomisation but before the intervention. At 24 weeks, 201 out of 230 participants (87.4%) completed the primary outcome and 216 out of 230 participants (93.9%) completed the PROMs. The treatment groups had similar participant characteristics. At 24 weeks, there was no difference in work LSI (mean difference –3.872; 95% confidence interval –10.454 to 2.710;p = 0.231), ATRS, pain or goal attainment between PRP- and placebo-injected participants. There were no differences between the groups in any PROM at any time point or in complication rates, including re-rupture and deep-vein thrombosis. There was no correlation between work LSI and platelet activation in PRP, or erythrocyte, leucocyte or platelet counts in whole blood or PRP. Biopsies showed similar cellularity and vascularity between groups.ConclusionsThis trial design and standardised PRP preparation gives the first robust RCT evidence about PRP’s role in managing ATR, which suggests that PRP offers no patient benefit. Equally robust evidence to investigate PRP application in tendon and soft tissue injuries is required. The 24-month follow-up will be completed in April 2020.Trial registrationCurrent Controlled Trials ISRCTN54992179.FundingThis project was funded by the Efficacy and Mechanism Evaluation programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership. The trial was supported by the NIHR Biomedical Research Centre, Oxford, and the NIHR Fellowship programme.

Variability of the Composition of Growth Factors and Cytokines in Platelet-Rich Plasma From the Knee With Osteoarthritis

To investigate the composition and concentration of growth factors and cytokines in platelet-rich plasma (PRP) with knee osteoarthritis and to explore the association of the concentration of growth factors and cytokines with the platelet count of PRPs. Patients who visited outpatient clinic with symptomatic knee osteoarthritis (Kellgren-Lawrence grades 1 to 3) and had no blood dyscrasia were enrolled from October 2014 to March 2015. PRPs were obtained using a commercial system. Concentrations of growth factors and cytokines were measured with an enzyme-linked immunosorbent assay. Anabolic factors (platelet-derived growth factor [PDGF]-AA, -BB, and -AB, transforming growth factor-β, vascular endothelial growth factor [VEGF], epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], and insulin-like growth factor 1), catabolic factors (interleukin [IL]-1β and matrix metalloproteinase 13), and catabolic blockers (IL-1 receptor antagonist) were included. The degree of variation was determined by coefficient of variation (CoV). 105 patients were included. Growth factors and cytokines showed wide variation. bFGF showed the highest variation (CoV 78.45), and transforming growth factor-β1 showed the lowest variation (CoV 5.30). Platelet count in PRP showed a positive correlation with PDGF-BB and -AB, and VEGF (r= 0.270, P= .005; r= 0.231, P= .018; and r=0.200, P= .041, respectively) and was negatively correlated with IL-1β (r= -0.220, P= .025). Growth factors and cytokines in PRPs obtained from patients with knee osteoarthritis show a wide variation; the highest variation was shown in bFGF. Platelet counts associated positively with PDGF-AB and -BB and VEGF and negatively with IL-1β. This information leads to the concept that variation and association of specific factors needs to be taken into consideration for future investigations of PRPs in clinical application in patients with knee osteoarthritis.

Effects of autologous platelet-rich plasma in the repair of soft tissue defects of rabbits with free flap

Objective: To explore the effects of autologous platelet-rich plasma (PRP) in the repair of soft tissue defects of rabbits with free flap. Methods: Thirty 6-month-old New Zealand white rabbits, male and female unlimited, were used to harvest blood from the heart. PRP was prepared by Aghaloo method, then free flap model with size of 5 cm×3 cm was reproduced on each ear of the rabbit. According to the random number table, one ear of each rabbit was recruited to PRP group, and the other ear was recruited to normal saline group. The base of flap on rabbit ear in PRP group was evenly spread with 1.0 mL autologous PRP, and equivalent volume of normal saline was applied to that in normal saline group. Then, the flap was replanted in situ. On post surgery day (PSD) 2, 3, 5, 7, and 14, 6 rabbits in each group were taken. The survival of flap was observed and recorded. The morphology of the basal tissue of flap was observed by hematoxylin-eosin staining. The expressions of CD31 and α smooth muscle actin (α-SMA) in the basal tissue of flap were detected by immunofluorescence method. Another 6-month-old male New Zealand white rabbit without making flap under the same experimental conditions was used for harvesting whole blood and preparing PRP. Then blood platelet count in whole blood and PRP was determined, and the content of vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) was detected by double-antibody sandwich enzyme-linked immunosorbent assay. Data were processed with analysis of variance of factorial design, paired sample t test, and Bonferroni correction. Results: (1) On PSD 2, the flaps of wounds of rabbits in PRP group were reddish and adhered well to the basal tissue; the flaps of wounds of rabbits in normal saline group were dark red and poorly attached to the basal tissue. On PSD 3, the flaps of wounds of rabbits in PRP group were ruddy and closely adhered to the basal tissue; the flaps of wounds of rabbits in normal saline group were scattered in the plaque-like dark red and generally attached to the base. On PSD 5, the flaps of wounds of rabbits in PRP group were reddish and closely adhered to the basal tissue, and the flaps were alive; while flaps of wounds of rabbits in normal saline group were rosy and closely adhered to the basal tissue. On PSD 7, the surface of flaps of wounds of rabbits in PRP group was covered with a medium amount of rabbit hair. The color of flap was similar to that of the surrounding skin. The flaps of wounds of rabbits in normal saline group were generally attached to the base, and the surface was only covered with a small amount of fluff. On PSD 14, the incisions were healed well in PRP group, while small wounds in normal saline group were not healed. (2) On PSD 2, inflammatory cell infiltration was observed in flaps of wounds of rabbits in both groups. On PSD 3, the flaps of wounds of rabbits in PRP group showed neovascularization, with less interstitial hemorrhage; while there were less neovascularization in the flaps of wounds of rabbits in normal saline group. On PSD 5, a medium number of inflammatory cell infiltration and a small amount of new microvessels were observed in flaps of wounds of rabbits in normal saline group. Many fibroblasts, a small amount of inflammatory cells, and scattered new microvessels were observed in flaps of wounds of rabbits in PRP group. On PSD 7, the number of new microvessels in normal saline group was significantly lower than that in PRP group. On PSD 14, the new microvessels in the flaps of wounds of rabbits in PRP group gradually matured, and a large number of fibroblasts distributed around them. Some of the newly formed microvessels in the flaps of wounds of rabbits in normal saline group were mature, and the healing was slower than that of PRP group. (3) On PSD 2, 3, 5, 7, and 14, the expressions of CD31 and α-SMA in the basal tissue of flaps of wounds of rabbits in PRP group were significantly higher than those in normal saline group (t=10.133, 5.444, 9.450, 6.986, 8.394, 14.896, 10.328, 9.295, 13.902, 10.814, P<0.01). (4) The platelet count in activated PRP of rabbits was (2 863±962)×10(9)/L, which was significantly higher than (393±49)×10(9)/L in whole blood (t=7.690, P<0.05). (5) The content of VEGF and TGF-β in activated PRP of rabbits was (564.3±3.2) and (1 143±251) pg/mL, which was significantly higher than (99.7±0.4) and (274±95) pg/mL in whole blood, respectively (t=287.390, 9.648, P<0.05 or P<0.01). Conclusions: PRP of rabbits contains high concentrations of VEGF and TGF-β. Therefore, PRP can effectively promote microvascular regeneration in free flap tissue and accelerate the survival of free flap.

Platelet yield and yield consistency for six single-spin methods of platelet rich plasma preparation

Single-spin methods of preparation of platelet-rich plasma are used widely in private practice, yet they have not been extensively studied and compared. The use of platelet-rich plasma (PRP) by the private practitioner can be facilitated by efficient and predictable PRP preparation. The primary purpose of this study was to study common methods of single-spin PRP preparation to determine their efficiency and variability. Six single-spin methods of PRP production from whole blood were analyzed. The primary measures were mean yield and standard deviation as the quotient of total platelet count in PRP produced divided by total platelet count in whole blood utilized. Each sample was analyzed in triplicate and the results were averaged. Secondary measures included red blood cell count (RBC) and white blood cell count (WBC), concentration ratio, and variable cost per million platelets produced. Sixty-four volunteers provided samples from 30 June 2017 to 30 September 2018. Seventeen to twenty six samples were utilized to assess each method. Yields for the six preparation methods (PMs) varied from 53(±18)% to 72(±13)%. Differences were observed for WBC count (1.8 to 14), Hematocrit (0.8 to 32), platelet concentration (568 to 1062), and variable cost per billion platelets produced ($1.55 to $44.31). All six methods evaluated provided a platelet yield of more than 50%, although two methods were less efficient than the others. Two methods were able to produce leukocyte-poor PRP. Variability was moderate across all methods, suggesting that estimation of platelet yield should be feasible from a baseline platelet count for all methods.

Application of standardized platelet-rich plasma in elderly patients with complex wounds.

In recent years, autologous platelet-rich plasma (PRP) derivatives have been used widely in the regeneration and repair of tissue, but a standard definition and preparation method for PRP are lacking. We developed a standardized method using platelet indices as quality-control indicators for PRP preparation. Twenty-one elderly patients (9 males, 12 females) with complex wounds were treated with standardized platelet-rich plasma (S-PRP). The platelet count in PRP after the second centrifugation was 1,069-1,436 × 109 /L. We adjusted the platelet concentration in PRP after a second centrifugation to 1,000 × 109 /L according to a formula using platelet-poor plasma (PPP). The standardized preparation method that we developed gave S-PRP with a relatively uniform platelet concentration. The wounds of 21 patients showed accelerated healing after S-PRP treatment, and there were no obvious side effects during treatment. These data suggest that our preparation method of S-PRP, using platelet indices as quality-control indicators with platelet count of 1,000 × 109 /L could be used for the treatment of complex wounds in the elderly. The preparation method of S-PRP proposed in the present study may be a simple and effective method of PRP quality control.

Growth factor levels in leukocyte-poor platelet-rich plasma and correlations with donor age, gender, and platelets in the Japanese population

BackgroundClinical application of platelet-rich-plasma (PRP) has been accelerated to investigate early recovery from various musculoskeletal conditions. It involves the promotion of tissue damage repair through the action of multiple growth factors at physiological concentrations. The composition of PRP differs based on many factors, which may include age and gender. Therefore, we analyzed correlations between age, gender, and platelet counts in PRP with growth factors in Japanese subjects.MethodPeripheral blood was drawn from 39 healthy volunteers between 20 and 49 years of age (age, mean ± standard deviation = 33 ± 8.7 years; gender ratio, male:female = 19:20; BMI, mean ± standard deviation = 22 ± 4.0) and prepared through centrifugation (volume, 6 mL per sample). After being activated with CaCl2, the supernatant was stored. The mean platelet count in PRP was 41.4 ± 12.2 × 104/μL. PRP concentration rate (i.e., PRP/peripheral platelet counts) was 1.8 ± 0.4 times. Growth factor levels (platelet-derived growth factor-BB, transforming growth factor-β1, vascular endothelial growth factor, epidermal growth factor, fibroblast growth factor, insulin-like growth factor-1, and hepatocyte growth factor) were measured using enzyme-linked immunosorbent assay (ELISA), and correlations with age, gender, and PRP platelet counts were statistically analyzed by calculating Spearman’s rank correlation coefficients (r).ResultsAge was negatively correlated with platelet-derived growth factor-BB and insulin-like growth factor-1 (r = − 0.32, − 0.39), and gender had no influence on growth factors. Platelet counts in PRP positively correlated with platelet-derived growth factor-BB, transforming growth factor-β1, epidermal growth factor, and hepatocyte growth factor (r = 0.39, 0.75, 0.71, and 0.48, respectively).ConclusionsThis clinical study shows a significant variation of PRP among individual patients and that this variation is influenced by the age and the platelet counts of the subjects. Our data demonstrate that patient characteristics account for the differences in PRP physiological activity.

A Study to Compare the Efficacy of Platelet-rich Plasma and Minoxidil Therapy for the Treatment of Androgenetic Alopecia.

Background:Androgenetic alopecia (AGA) is the most common cause of hair loss in men with limited treatment options. Platelet-rich plasma (PRP) therapy is one of the newer treatment options in the management of AGA which has shown promising results.Aims and Objectives:This study was aimed at comparing the clinical efficacy of PRP therapy with minoxidil therapy.Materials and Methods:In the study, patients were randomized into two groups – Group A (given PRP therapy) and Group B (given minoxidil therapy). Both groups were followed up over a period of 6 months, and final analysis was done with the help of global photography, hair pull test, standardized hair growth questionnaire, patient satisfaction score; in addition, a comparison of platelet counts in PRP was done, to know that if a clinical correlation exists between platelet concentration and clinical improvement. A total of 40 patients clinically diagnosed with AGA were enrolled in the study with 20 patients in each group. Four patients from Group A (PRP) and six patients from Group B (minoxidil) could not complete the treatment for 6 months and were eventually excluded.Results:At the end of 6 months, 30 patients were evaluated to compare the efficacy of intradermal PRP and topical minoxidil therapy. On global photography, Group A (PRP) was found to have a comparatively better outcome than Group B (minoxidil). In hair pull test, hair growth questionnaire, and patient satisfaction score, Group A was found to be better than Group B. Mean platelet count at baseline was 3.07 ± 0.5 lac/mm, 3 while platelet count in final PRP prepared was 12.4 ± 1.7 lac/mm, and patients with a higher platelet count in PRP had a much better clinical improvement compared to patients with a low platelet count in PRP. Side effects with PRP therapy were minimal with better results which may improve the compliance of the patient.Conclusion:PRP therapy can be a valuable alternative to topical minoxidil therapy in the treatment of AGA.

Effect of autologous PRP on wound healing in dental regenerative surgeries and its correlation with PDGF levels.

INTRODUCTION:Autologous platelet-rich plasma (PRP) is the fraction of blood plasma, with increased concentration of platelets, from baseline serum level. Growth factors (GFs) in PRP expedite the soft tissue and bony healing. However, estimation of their levels and role in healing had not been studied extensively. This study gives an insight to the quantification of platelet-derived GF-BB (PDGF-BB) present in PRP and its correlation with the clinical wound healing and bone regeneration.AIMS:This study aims to quantify PDGF-BB levels in PRP with its subsequent correlation with healing in dental regenerative surgeries.SETTINGS AND DESIGN:This was an experimental study including patients undergoing various dental regenerative surgeries.SUBJECTS AND METHODS:Autologous thrombin-activated PRP in the form of PRP gel was used in study group (n = 39) whereas no such intervention was given in control group (n = 30). PDGF-BB quantification was done in PRP samples using enzyme-linked immunosorbent assay. Clinicoradiological evaluation of healing was done in both the groups.STATISTICAL ANALYSIS USED:Descriptive analysis, independent Z-test, Correlation regression analysis, and ANOVA.RESULTS:Mean platelet concentration achieved in PRP was 5.79 times the baseline count. Mean PDGF-BB concentration in PRP was 31.92 ± 10.47 ng/ml which significantly correlated (P < 0.05) with the PRP platelet count. Study group showed significant healing clinically (P < 0.05). Significant bone fill observed in study group at 3 and 6 months when compared to the baseline as well as control group. Furthermore, bone fill at 6 months showed linear correlation with PGDF-BB levels (r = 0. 80).CONCLUSIONS:PRP led to enhanced bone regeneration and soft-tissue healing with former being directly related to higher concentration of PDGF-BB.

Spectrophotometric determination of platelet counts in platelet-rich plasma

BackgroundPlatelet-rich plasma (PRP) is widely used in regenerative dentistry and other medical fields. However, its effectiveness has often been questioned. For better evaluation, the quality of individual PRP preparations should be assured prior to use. We proposed a spectrophotometric method for determination of platelet counts and validated its applicability using two types of PRP preparations.MethodsBlood samples were obtained from healthy male volunteers and pure PRP (P-PRP) and leukocytes-rich PRP (L-PRP) were prepared using the double-spin method. In serial dilutions, platelet counts in P-PRP and L-PRP were determined using an automated hematology analyzer and a compact spectrophotometer. For validation, P-PRP and L-PRP independently prepared by three well-trained operators were used for comparison of the calculated and measured platelet counts.ResultsIn the two types of PRP samples evaluated, platelet counts were almost equal and greater amount of both white blood cells (WBCs) and red blood cells (RBCs) were included in L-PRP preparations. The calibration curve obtained from serially diluted P-PRP showed a strong correlation (R2 = 0.995), whereas that of L-PRP was relatively weaker (R2 = 0.975). In validation testing, the scatter plot of the calculated platelet counts versus the measured values showed a strong correlation in P-PRP (R2 = 0.671), whereas that of L-PRP showed a much weaker correlation (R2 = 0.0605).ConclusionsThis method can precisely determine platelet counts in PRP preparations when the inclusion of WBCs or RBCs is minimized. Therefore, we recommend that clinicians use this method for quality assurance of individual PRP preparations.

PDGF-AB rich-trombocyte lysate supplementation from breast cancer patients increased the proliferation of breast cancer stem cells

Background: Thrombocytosis in breast cancer (BC) patient was thought to play a role in the invasiveness of breast cancer stem cells (BCSCs). Modification of tumor microenvironment was proposed to increase the efficacy of anticancer therapy. This study was aimed to analyze the effect of platelet lysate (PL) as well as its PDGF-AB content as a tumor microenvironment on (CD24-/CD44+) BCSC proliferation.Methods: This was an experimental study that treated culture of BCSCs with PL from breast cancer (BC) patients or healthy donors. Venous blood from all subjects were subjected to prior hematology test and then processed to obtain platelet rich plasma (PRP). Platelet counts in PRP were determined. PRP was processed to obtain PL. PDGF-AB contents in PL were measured. PL at concentrations of 0.01% (v/v) was supplemented into DMEM-F12 medium and used for culturing BCSCs (CD24-/CD44+ cells). After 48 hours, total cell count, population doubling time (PDT), and cell viability were calculated and their correlation with platelet count and PDGF-AB levels were analyzed.Results: BC patients (n=5) had higher platelet counts and PDGF-AB levels in PL compared to healthy donors (n=15), (p=0.02). PL from BC patients could stimulate the proliferation of BCSCs higher than healthy donors (p&lt;0.001) and showed lower PDT value (p=0.001). Cell proliferation and PDT showed strong correlation with PDGF-AB level. This observation suggests that PDGF-AB has a role on BCSCs proliferation. PL showed no effect on BCSCs viability.Conclusion: Breast cancer patient platelet lysate stimulated BCSC proliferation.

Inhibitory Synergistic Effect of 830nm LASER Radiation and Serum‐Rich in Growth Factors on Viability of Human Fibroblasts In Vitro

LASER radiation (LR) at the wavelength (λ) of 830 nm and serum‐rich in growth factor (SRGF) derived from platelet rich plasma (PRP) are used clinically as biostimulating agents in tissue repair. In contrast, the influence of the synergistic effects of these therapeutics tools on cellular activity are unknown. For this purpose, a protocol was designed to evaluate, in vitro, the isolated and synergistic biomodulatory action of 830 nm LR and SRGF on viability of human fibroblasts (GM07492 cell line). Samples of blood from a male volunteer were submitted to a double centrifugation protocol (400g‐10min./700g‐17min.) to obtain the PRP, posteriorly activated with human thrombin solution (10,000 IU) and calcium chloride (10% m/v) to obtain SRGF. In order, to analyze the influence of LR on the wavelength (λ) of 830nm, continuous emission mode, energy density of 0.3J/cm2 and irradiance of 18mW/cm2 were established. Cells were grown in 24 well plates (3×104 cells/well) in Dulbecco's Modified Eagle Medium (DMEM), supplemented with fetal bovine serum (FBS‐2.5%), SRGF (2.5%) or combination of both. The cell viability rate was determined in each of these culture conditions, associated or not to LR, using the MTT‐Formazan colorimetric method. The analyzes were performed 24 hours after irradiation and the viability (triplicate) expressed in percentage in relation to the control condition (FBS‐2.5%). The data were submitted to analysis of variance (ANOVA) supplemented by Fisher's post‐test, considering a significance level of 5% (p≤0.05). The platelet counts in PRP showed a concentration of 1.7×106/μL of plasma. The results regarding cell viability demonstrated that SRGF exerted a biostimulating effect on cells, considering a 27% increase in viability rate when used alone and 116% when associated with FBS. LR at λ also exerted a biostimulating effect on cells cultured in the control condition and with SRGF alone, providing a 164 and 142% increase in cell viability rate, respectively. In contrast, the synergistic action of radiation and SRGF associated with FBS determined a bioinhibitory effect in the cells, considering the reduction of 53% in the viability rate in relation to the cells in the same culture conditions and without the radiation. The association of these availability sources of growth factors (GF) were used to simulate, in vitro, the therapeutic use of PRP, providing more growth factors into the repair site. The results allow us to conclude that both 830 nm LR and SRGF have an in vitro biostimulating effect on viability of human fibroblasts when they act in isolation and that the synergistic influence of SRGF and LR promote a significant bioinhibitory cellular effect that could compromise the therapeutic benefits sought in a tissue repair process when they are used.Support or Funding InformationCoordenação de Aperfeiçoamento de Pessoal de Nível SuperiorThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of pH and concentration of sodium citrate anticoagulant on platelet aggregation measured by light transmission aggregometry induced by adenosine diphosphate

The 2013 ISTH-SSC guidelines for the standardization of light transmission aggregometry (LTA) were largely based on expert consensus, as studies directly comparing LTA methodologies were lacking. We experimentally tested the cogency of ISTH-SSC recommendations pertaining to use of anticoagulant, in particular whether: (1) buffered citrate (BC) is preferable to unbuffered citrate (C); (2) the two recommended concentrations of sodium citrate (109 and 129 mM) are equivalent in terms of platelet aggregation (PA). Blood from 16 healthy volunteers was collected into BC and C (109 and 129 mM). PA was measured by LTA in platelet-rich plasma (PRP) stimulated by adenosine diphosphate (ADP) (2 μM) immediately after PRP preparation and up to 4 hr after blood collection; pH and platelet counts in PRP were measured in parallel. pH in PRP increased with time up to about 8 for all anticoagulants, although it was lower in BC than in C at all times. In BC, PA was lower at 45 min, but equivalent at all other times. PA was higher and more stable in sodium citrate 109 mM than in 129 mM at all times. The extent of PA did not change for up to 2 hr after blood collection, and subsequently dramatically decreased. In contrast with ISTH-SSC recommendations, (1) BC does not show advantages compared to C; (2) 109 mM citrate is preferable to 129 mM, because it better supports PA; and (3) LTA studies should be completed within 2 hr of blood collection, instead of the recommended 4 hr.

Growth Factors in the Platelet-Rich Plasma

PRP is an useful bioproduct to tisular regeneration. The aim of study was evaluate the concentration of growth factors: PDGFBB (platelet-derived growth factor), EGF(epidermal growth factor) and VEGF (vascular endothelial growth factor) present in the Platelet-rich plasma (PRP) in subjects treated with drugs which inhibit platelet aggregation as acetylsalicylic acid (ASA) and clopidogrel before and after administration. We determined by ELISA PDGFBB, EGF and VEGF levels in PRP, Platelet Poor Plasma (PPP), lysate and exudate from 32 healthy subjects before and 24 hours after ingesting acid Acetyl salicylic acid (ASA) and clopidogrel as a single dose. The PRP and PPP were obtained by the method of Anitua by single centrifugation method. To analyze the results of student test and Pearson correlation was applied, with statistical significance level of p &lt; 0.05. PPP and exudate (Clopidogrel: p &lt; 0.001), PRP (Clopidogrel: p &lt; 0.01) statistically significant differences for PDGFBB in PPP (p &lt; 0.01 AAS) were found, and for VEGF in lysate (ASA and Clopidogrel: p &lt; 0.05). No significant difference was found for EGF. Only was no correlation between baseline values of EGF in the ASA group and the respective PRP platelet count (r = 0.726). The results show that the average basal values of the three growth factors measured were considered particularly high in the PRP and lysate, showing the significant decrease for PDGFBB after antiplatelet therapy, especially of Clopidogrel and a significant increase for the VEGF only for the lysate. Although the behavior of three different soluble mediators was different to antiplatelet agents, the observed changes support the conclusion that a single dose of these drugs not markedly affect the secretion and availability of the three growth factors measured in various platelet derived obtained.

Evaluation of an automated light transmission aggregometry

Light transmission aggregometry (LTA) is the “gold standard” for platelet function assessment, but it is time-consuming and labor intensive. Recently, an automated platelet aggregation method has been developed on a routine coagulation analyzer (Sysmex CS-2100i). In this study, the performances of CS-2100i including repeatability, correlation with a reference aggregometer (Chrono-log Model 700), and the threshold limitation of platelet counts in platelet-rich plasma (PRP) were evaluated for clinical use. The agonists were adenosine diphosphate (ADP), arachidonic acid, collagen, epinephrine, and ristocetin. The platelet concentration of PRP was adjusted with platelet-poor plasma (PPP) and physiological saline (PS). The CS-2100i showed an excellent repeatability and a strong correlation with the Chrono-log 700 in performing platelet aggregation, and its threshold limitation of platelet counts in PRP was 80 × 109/L. PPP had an inhibitory impact on platelet aggregation induced by ADP, arachidonic acid, collagen or epinephrine; while PS had an inhibitory impact on ristocetin-induced aggregation. PS should be used to adjust PRP for ADP-, arachidonic acid-, collagen-, or epinephrine-induced aggregation; while PPP was recommended for ristocetin-induced aggregation. The CS-2100i showed an excellent repeatability, a strong correlation with Chrono-log 700, a lower platelet count requirement, a shorter turnaround time for samples, the advantage of being a walk-away technology, and the ability to perform a highly standardized platelet function assessment.

Influence of calcium salts and bovine thrombin on growth factor release from equine platelet-rich gel supernatants.

To compare five activation methods in equine platelet-rich plasma (PRP) by determination of platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) concentrations in platelet-rich gel (PRG) supernatants. Platelet-rich plasma from 20 horses was activated by calcium chloride (CC), calcium gluconate (CG), bovine thrombin (BT), and their combinations, BTCC and BTCG. Both growth factor concentrations in PRG supernatants were measured by ELISA and compared with plasma and platelet lysates (PL) over time. Growth factor concentrations were significantly lower in plasma and higher for all PRG supernatants. Platelet lysates contained a significantly lower concentration of PDGF-BB than PRG supernatants and a significantly higher concentration of TGF-β1 than PRG supernatants. Clots from PRP activated with sodium salts were more stable over time and had significant growth factor release, whereas CC produced gross salt deposition. Significant correlations were noticed for platelet with leukocyte concentrations in PRP (rs: 0.76), platelet counts in PRP with TGF-β1 concentrations in PRG supernatants (rs: 0.86), platelet counts in PRP with PDGF-BB concentrations in PRG supernatants (rs: 0.78), leukocyte counts in PRP with TGF-β1 concentrations in PRG supernatants (rs: 0.76), and PDGF-BB concentrations with activating substances (rs: 0.72). Calcium gluconate was the better substance to induce PRP activation. It induced growth factor release free from calcium precipitates in the clots. Use of BT alone or combined with calcium salts was not advantageous for growth factor release.