Variability in Platelet-Rich Plasma Preparations Used in Regenerative Medicine: A Comparative Analysis

Table 1

Includes various methods used to prepare PRP. Includes type of study, preparation method, characterization of products, any additives used, and the results.

S. No	Articles	Type of study	Injectable product/product type used	PRP Preparation Method	Product Characterization	Additives used/ Additional procedures	Results
1	Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential	In Vitro	PRP	Leukodepleted platelet-rich plasma (PRP) was collected by apheresis from four donors using an automated blood collection system (Mobile Collection System MCS+, Haemonetics Corp., Boston, MA, USA), according to the manufacturer’s instructions. Acid Citrate, Dextrose Solution A (ACD-A) was used as an anticoagulant.	1.Platelet count, 2. fibrinogen levels,3. 28 growth factors and 8 GF receptor concentration, 4. Effect on in vitro proliferation, 5. differentiation of osteoblast cells.; Effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation.	Platelet activation is conducted by single and double freeze-thaw cycles.	Mean PLTs content in PRP samples 4.6-fold higher.; Similar trends of GF in all 4 samples, their relative concentrations were different.
2	Implementation of a closed platelet-rich-plasma preparation method using the local blood bank infrastructure at the Principality of Asturias (Spain): back to basic method and a demographics perspective after 1 year.	In Vitro	PRP	Closed system PRP preparation method using the infrastructure of a certified blood bank. 1.Extraction of 150mL blood. Transported in butanediol plates (constant temperature 20-24ºC). 2.First centrifugation (425g, 5 min, acc6, no brake, 22oC), to separate plasma fraction containing platelets with a press Compomat G5 (Fresenius) into a new bag. 3.Second centrifugation (1328g, 12 min, acc9, break 4, 22oC), to separate approximately 30mL of PRP into a new bag from the remaining platelet poor plasma. 4.The PRP is allowed to rest for 4 hours and kept overnight agitating at room temperature. 5.The PRP is then distributed into 3 pediatric transfusion bags (10 mL each). 6.Aliquots are frozen at -40ºC to allow platelet lysis and cargo release and can be stored for 2 years. The PRP is distributed to the petitioner hospital on dry ice. 7.The PRP is prepared within 24 hours of extraction and stored frozen, and the cold chain is maintained until its use.	They prepared 553 PRP units in 1 year.; 1. The Multiplex Technology was used to measure concentration of several relevant growth factors like EGF, HGF, PDGF-BB, VEGF-A, VEGF-D, FGF-23.; 2. PRP validation done by measuring WBC counts and platelet counts in whole blood and PRP across various samples.	Thawed PRP and using CaCl2.	Individual variations in platelet derived GFs were noted amongst donors and the concentration of factors amongst the three frozen aliquots derived from a single donor remained constant.
3	The effect of the anticoagulant on the cellular composition and growth factor content of platelet-rich plasma	In Vitro	Freshly prepared PRP	Single phlebotomists used T-Lab (T-Biotechnology, Bursa, Turkey) standard 0.9 ml 3.8% SC containing PRP tubes and PRP tubes without anticoagulants. Approximately 26 ml’s of venous blood was obtained from each participant via 21G butterfly needle from antecubital vein, and the first 2 ml of blood was discarded to avoid platelet activation during blood collection. An additional 3 ml blood was collected from all volunteers for complete blood cell analysis. Two T-Lab swing rotor centrifuges were used for PRP preparation. PRP volumes obtained were recorded with the use of a 10- mL graduated pipette. The platelet, RBC, and WBC concentrations of the fresh PRP products from each protocol and whole blood were determined using hematology analyzer.; Protocol 1: Approximately 7 ml blood was collected into the tubes prefilled with 0.9 ml SC, then, they were centrifuged at 1000 g for 5 min.; Protocol 2: Approximately 8 ml blood was collected into the tubes without anticoagulant. Then, 0.5 ml SC was added. Immediately after blood collection, they were centrifuged at 1000 g for 5 min.; Protocol 3: Approximately 8 ml blood was collected into the tubes without anticoagulant. The first 2 ml of blood were discarded to avoid early activation of the platelets. Immediately after blood collection, they were centrifuged at 1000 g for 5 min.	The PRP’s were compared regarding cellular content (rbc, wbc, platelets), capture efficiency of platelets (CE), concentrations and total doses of fresh studied vascular endothelial growth factor (VEGF), platelet derived growth factor -BB, (PDGF-BB), transforming growth factor b1 (TGF-b1) levels.	No additives/activators	CE, total platelet count was highest in protocol 1. The white blood cells (WBC) and VEGF were highest in protocol 3. The highest total TGF-b1 and total PDGF levels were obtained with protocol 1, while the highest total VEGF levels were obtained with protocol 3.
4	An in vitro long-term study of cryopreserved umbilical cord blood-derived platelet-rich plasma containing growth factors —PDGF-BB, TGF-β, and VEGF	In Vitro	UCB-PRP cryopreserved for 3 years and thawed.	UCB was collected by puncturing the umbilical vein after the expulsion of the placenta. Immediately after collection, UCB was mixed with ACD-A (Terumo Co., Ltd., Tokyo, Japan) for anticoagulation. Subsequently, the mixture was centrifuged twice (2,400 rpm, 10 min; 3,600 rpm, 10 min) at 20 °C to obtain UCB-PRP and UCB-derived platelet-poor plasma. UCB-PRP was cryopreserved at −80 °C until the time of use. UCB-PRP, thawed at room temperature just before use, was randomly allocated to the experiments. Immediately after collection, the concentration rates of UCB and UCB-PRP per 1 mL of platelet count were calculated. The concentrations of growth factors (i.e., PDGF-BB, TGF-β1, and VEGF) which were contained in UCB at baseline, 3 months, and 3 years of cryopreservation, were determined quantitatively according to enzyme-linked immunosorbent assay.	1. Baseline, 3 months, and 3 years.; 2. Measured PDGF-BB, TGF-β1, and VEGF) using ELISA.; 3. Measured platelet concentration	No information on additives/Activators.	1.growth factors in cryopreserved UCB-PRP were markedly elevated compared to those found in UCB at baseline.; 2.Cryopreserved UCB-PRP possibly and advantageously induced the osteoblastic differentiation of UC-MSCs.
5	Stabilization of porous chitosan improves the performance of its association with platelet-rich plasma as a composite scaffold.	In vitro	Activated PRP	1. Preparation according to Perez et al.,; whole blood (WB) was collected in 3.5 mL vacuum tubes (Vacuette®, Campinas, SP, Brazil) containing sodium citrate 3.2% (w/v) as an anticoagulant.; 2. centrifuged in a Rotina 380R centrifuge (Hettich® Zentrifugen, Tuttlingen, Germany) at 100×g for 10 min at 25 °C.; 3. The upper layer was collected as P-PRP.; 4. Concentration of platelets, WBCs, and RBCs in WB and P-PRP was determined using the ABX Micros ES 60 hematology analyzer (HORIBA ABX Diagnostics, Montpellier, France).; 5. Activated P-PRP (aP-PRP) prepared using autologous serum (Ser) and 10% (w/v) CaCl2 solution as agonists in the following proportions: agonist/P-PRP = 20% (v/v); Ser/CaCl2 volumetric ratio = 9. (Autologous serum was prepared by collecting 5 mL of WB in tubes without anticoagulant. After 30 min of clot formation, WB was centrifuged at 2000×g for 10 min).	The scaffolding was characterized by porosity, non-cytotoxic, pore size and young's modulus.; PDGF-AB and TGF-β1 were measured using ELISA kits.	Autologous serum and CaCl2.	SPCHTs showed controlled release of the growth factors TGF-β1 and PDGF-AB. All the variations of stabilized scaffoldings showed cell differentiation more than non-stabilized ones.
6	Clinical-grade quality platelet-rich plasma releasate (PRP-R/ SRGF) from CaCl2-activated platelet concentrates promoted expansion of mesenchymal stromal cells.	In Vitro	PRP-R (platelet-rich plasma releasate) /SRGF (supernatant rich in growt factors)	1.Leucocyte-depleted platelet-rich plasma (PRP) was obtained by plasma platelet apheresis from healthy donors.; 2.PRP was manipulated in aseptic conditions (validated clean room).; 3.Platelet activation was performed in PRP by addition of CaCl2.; 4.Supernatants of centrifuged samples (PRP-R/SRGF) were separated into aliquots and stored at -80°C without the addition of heparin.; 5. Microbiology analysis was done to rule out bacterial or fungal contamination.	1. Platelet concentration not available.; 2. PDGF-AA, PDGF-AB, PDGF-BB, TGF-b, FGF, EGF, IGF-1 and VEGF levels were measured by ELISA.	CaCl2 used for activation.	PRP-R/SRGF was more active than FBS to expand BM- and AT-derived; MSCs. PRP-R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T-cell proliferation.
7	The Number of Platelets in Patient’s Blood Influences the Mechanical and Morphological Properties of PRP-Clot and Lysophosphatidic Acid Quantity in PRP	In vitro	PRP	1. Venous blood from healthy male and female volunteers aged between 20 and 60 years; nonsmokers; and absence of chronic hematologic, neoplastic, and/or infectious diseases (HIV+, HCV+, and HBV+).; 2.Samples of collected blood to obtain a platelet count.; 3. Samples were then centrifuged at 2500 rpm for 8 min at 25 ◦C.; 4. The PRP portion was removed as two fractions of equal volume: the more superficial one, called fraction 1 (PRP-F1), and the part nearest to the leukocyte fraction, called fraction 2 (PRP-F2). Platelet counting of PRP-F1 (F1) and PRP-F2 (F2) was performed before placing F2 into sterile vials and activating it according to Endoret® kit using 10% CaCl2 at 37 ◦C for 1 h to create the platelet concentrate (F2-clot).; 5. Moreover, the in vitro activity of the PRP clots on proliferation and migration of osteoblast-like cells was assessed together with LPA quantification that was done on PRP-F1 (F1), PRP-F2 (F2), and Endoret®-activated PRP-F2 (F2-clots). Plasma concentrates (F2-clots) prepared from PRP from patients with low platelet number (group A) and PRP from patients with high platelet number (group B) were compared.	1. Platelet number; 2. LPA was quantified before and after PRP fractioning and; activation.	CaCl2 used for activation	There was significantly higher plasma level of LPA in patients with a higher platelet concentration (group B) in; comparison to those in group A (p < 0.05). This different concentration was evidenced in PRP but not in the clots. Higher level of LPA in PRP from patients richer in; platelets should be considered as responsible for the higher hOB activity in bone regeneration.
8	The production method affects the efficacy of platelet derivatives to expand mesenchymal stromal cells in vitro.	In Vitro	Platelet lysate and SRGF.	1. Platelet apheresis were collected from 15 donors, transferred to 50-ml tubes (Falcon, Corning MA, USA) and stored at −80 °C. After 2 cycles of freezing/thawing the aliquots were centrifuged at 1600×g for 15 min at room temperature and the supernatants were collected, pooled, filtered using a 70 μm cell strainer (Falcon, Corning MA, USA) and finally stored at −20 °C until use.; 2. Preparation of PR-SRGF: From the apheresis sample, platelet activation was performed by adding CaCl2 at the final concentration of 0.04 M and after incubation at 40 °C for approximately 60min until complete clot formation. Bags were centrifuged for 5 min at 2200×g and the SRGF collected and stored at −80 °C	1. Concentration of PDGF-AB, PDGF-AA, PDGF-BB, EGF, VEGF, FGF-basic, IGF-1, TGF-β1 were quantified by using ELISA Kits.; 2. Platelet concentration.	Freeze/thawing & CaCl2.	The concentration of PDGF-AB, PDGF-AA, PDGF-BB in PR-SRGF resulted to be respectively 5.7×, 1.7× and 2.3× higher compared to PL. PR-SRGF promoted a higher BM-MSC proliferation rate compared to PL not altering BM-MSC phenotype. Colony forming efficiency of BM-MSC expanded in PR-SRGF showed a frequency of colonies significantly higher than cells expanded in PL. BM-MSC expanded in PL or PR-SRGF maintained their immunomodulatory properties against activated lymphocytes even if BM-MSC expanded in FBS performed significantly better.
9	Platelet-Rich Plasma Centrifugation Changes Leukocyte Ratios.	In Vitro	LR-PRP & LP-PRP	1. Gel-tube Method ~10cc of blood was spun at ~950g for ten minutes in a tube containing 1-2cc of a gel with a density slightly higher than platelets. The bottom 3cc of the plasma layer was removed as PRP.; 2. Double-syringe Method ~15cc of blood in an ACP double syringe (Arthrex ACP Double-Syringe System; Arthrex Inc., Naples, FL, USA) was spun at 300g for five minutes and the plasma layer was withdrawn as PRP.; 3.Machine Method 90-180cc of blood was placed into an Angel PRP machine (Angel® Concentrated Platelet Rich Plasma System; Arthrex Inc., Naples, Florida, USA) and processed with the setting set for a hematocrit of 4%. 4.Yellow-top Tube Method An ACD-A blood collection tube (BD Vacutainer ACD, catalog #364606; Becton-Dickinson, Franklin Lakes, NJ, USA) was filled with blood and spun at 1000g for ten minutes. The buffy coat and 1-2cc of the plasma layer just above it was extracted for PRP. 5.Single-syringe Method 15cc of blood was drawn into a syringe containing 1.5cc of sodium citrate solution and spun for ten minutes at 1000g. 0.6cc just below the buffy coat and 4cc above the buffy coat were removed as PRP.	1.Platelet count; 2. Leukocytes and granulocytes numbers.	None	There is a significant shift, increase in lymphocyte percentage and decrease in granulocyte percentage is evident across PRP preparation methods
10	Bone marrow concentrate and platelet-rich plasma differ in cell distribution and interleukin 1 receptor antagonist protein concentration	In Vitro	PRP and BMC	1. Blood (25 mL) was drawn into a syringe holding 4 mL acid citrate dextrose (ACD).; 2. Bone marrow was aspirated from the iliac crest into a 30-mL syringe holding 4 mL ACD. 1 mL was retained as the BMA sample for the study, and the rest was separated into two 60 mL samples and processed in two systems; Magellan® (BMC-A) (Arteriocyte Medical Systems Inc.) and SmartPrep® 2 (BMC-B; Harvest Technologies Corp., Plymouth, MA). All aspirations were performed by the same surgeon (JGK). All samples were processed within 24 h of collection.	1. Cellular concentrations of Platelets, rbc, wbc were assessed for all samples. 2.FGF-1, PDGF‑BB, VEGF, IL-1β, IL-6, IL-8, TNFα, IL-1ra, IFN-γ, TGF-β1, TGF-β2, 3 were measured using ELISA.	None	Colony-forming units were increased in both BMCs compared to BMA (p < 0.0001). Surface markers were consistent with MSCs. Platelet counts were not significantly different between BMC-A and PRP, but there were differences in leukocyte concentrations. TGF-β1 and PDGF were not different between BMC-A and PRP. IL1ra concentrations were greater (p = 0.0018) in BMC-A samples (13,432 pg/mL) than in PRP (588 pg./mL). The IL-1ra/IL-1β ratio in all BMC samples was above the value reported to inhibit IL-1β
11	Turn down - turn up: a simple and low-cost protocol for preparing platelet-rich plasma	In vitro	PRP	Turn Down-Turn Up PRP Protocol - Double Spin - Closed System: 1. Collect the desired volume (8.5 ml) of blood through peripheral venous access directly into a vacuum tube with acid citrate dextrose; (ACD) (1.5 ml).; 2. Equalize the remaining vacuum in the tube.; 3. Centrifuge the tube at 200 _x0001_g for 15 minutes with the tube cap facing down.; 4. Carefully remove the tube from the centrifuge and support the tube in the downward position without turning the tube.; 5. Under aseptic conditions, aspirate 3.5 ml of the hematic layer through the rubber cap.; 6. Turn the tube to an upright position (cap facing up).; 7. Centrifuge the tube at 1600 _x0001_g for 10 minutes with the lid facing up.; 8. Under aseptic conditions, aspirate 3.5 ml of the superior part of the material (platelet-poor plasma, PPP).; 9. Aspirate the desired amount of PRP (1-2 ml) from the lower part of the tube.	1.Platelet concentration was measured in whole blood and PRP.	None	Four methods obtained concentrations of platelets that were 1.15-, 2.07-, 2.18-, and 3.19-fold, respectively.; With the turn down-turn up technique, 4.17-fold (95% confidence interval (CI)
12	Production of platelet-rich plasma gel from elderly patients under antithrombotic drugs: Perspectives in chronic wounds care.	In Vivo	PRP and PRP gel	PRP gel preparation; 1. Venous blood: The 20-ml anticoagulated syringe was transferred to the PRP tube while the 10-ml non anticoagulated blood was transferred into the PRAS® Gel tube to obtain serum containing autologous thrombin.; 2. Centrifugation of both tubes was performed at 900G for 12 minutes.; 3. Through luer lock connection, PRP and autologous thrombin were collected from each single tube.; 4. One ml of PRP was kept for quality control including complete blood cell count, aggregometry and P-selectin expression.; 5. Remaining PRP and autologous thrombin were mixed in an 8-cm diameter glass cupule with CaCl2 in the following way: PRP was first added in the glass cupule, followed by 0,5 ml of CaCl2 and mixed for 10 seconds. Then, autologous thrombin was added and mixed for 10 seconds. From there, time formation for the gel was assessed visually every 30 seconds until complete gel formation.	1.Platelet concentration; 2. P-selectin expression; 3.rbc, wbc concentration; 4. Platelet aggregation in response to TRAP-6; 5.Coagulation profile; 6. Autologous Thrombin; 7. Fibrinogen	1. Autologous thrombin and Cacl2.	1. No significant difference was observed in the volume, composition (quantity of platelets, leukocytes, and red blood cells) and functionality of platelets from PRP except a higher ADP-induced P-selectin expression in healthy donors compared with elderly patients.; 2. Autologous thrombin characteristics were similar in the two groups. Concentrations of theoretical thrombin generated in the serum and in the gel were inversely correlated with the time of formation of PRP gel (r2 = 0.57, p = 0.012).
13	Enrichment of plasma in platelets and extracellular vesicles by the counterflow to erythrocyte settling.	In vivo	Platelet and EV rich plasma	Blood in the vacutainer is centrifuged at 300g for 5 min to obtain EPP (Erythrocyte poor plasma). This fraction is seperated and centrifuged again at 700 g for 17 minutes to obtain PVRP/PPP (Platelet & extravesicular rich plasma/Platelet poor plasma). Citrate as anticoagulant.	5 populations of particles were followed by Flowcytometry -- P1: attributed mainly to erythrocytes but containing also leukocytes, P2 and P2 (in larger and in smaller scale settings, respectively): attributed mainly to activated platelets and larger EVs, P3: subpopulation of particles with a weak side scattering signal, and Pa+Pb: attributed to smaller EVs and lipoproteins.	None	Study noted variations in the platelet concentration and EV’s in the suspension which were related to sample handling, number of centrifugations,processing time, temperature, ESR, centrifugation pull and volume of blood.