Commonly used procedures: Venous blood collected using a vacutainer tube, syringe, or commercial kits are commonly used to prepare PRP . Technologically advanced and equipped centers may use apheresis which gives leukocyte depleted PRP, which is a better quality PRP than venous blood derived PRP, which may have few leukocytes too . However, there is a rising discussion about the presence of number and type of WBC’s in PRP in some musculoskeletal studies, who argue that it is beneficial to have few WBC’s in PRP , on the contrary some others have stated that the Leukocytes should be avoided in PRP preparations because of their potential proinflammatory effect . Some centers worked with umbilical cord blood collection from the umbilical vein of fresh placenta and added anticoagulants like acid citrate-dextrose (ACD) before further processing . Large scale production of PRP using closed systems in blood banks have been used . Centrifugation is the main process used for density gradient-dependent separation of PRP from whole blood samples, unlike apheresis or large-scale blood bank operations. Effect of normal temperature vs temperature controlled, fixed angle vs horizontal rotors, single spin method vs double spin methods and use of anticoagulant on the fold of platelet yield/GF’s is well documented [5, 8]. In cases of frozen PRP, freeze-thaw cycle is considered to activate the PRP, as also the high-speed centrifugation (1000–10,000 G for a long duration) and has been considered to be damaging to the platelets, causing degradation of platelets and release of platelet derived factors . The non-activated PRP obtained from the previously mentioned methods is activated using Calcium chloride (CaCl2), fibrin and/or thrombin and autologous serum to release growth factors (In vitro), both in case of fresh PRP and freeze thawed PRP [8, 10, 12]. New engineering techniques such as biologic scaffoldings are also under analysis and development which would prolong the release of factors from platelets/PRP in vivo for sustained therapeutic effects .
Addressing the Variability
Procedure variability: The PRP preparation procedures through generations of evolution have experimented with various physical parameters to progress towards the development of a standardized method and the most efficacious PPR/PRP product. The goal is still in distant sight. We lack complete and transparent large-scale data . The major reason for variations in the data and its comparability is due to variable approaches in preparation methodology such as, the volume of blood collected for processing, method of collection (Apheresis vs Venous blood), source of sample (Venous blood vs Umbilical cord blood), different equipment, single centrifugation vs double centrifugation methods, normal temperature processing vs temperature controlled settings, automation vs manual aspects, studies using fresh PRP vs Freeze-thawed PRP, usage of anticoagulants, additives, etc. . Some authors believe that PRP products can be used without an activation agent because platelets are spontaneously activated upon exposure to dermal collagen and thrombin after in vivo injection [29,30].The usage of exogenous activating substance is controversial and it is important for Physicians to mandatorily mention the activation status, since different PRP activation agents are thought to influence the physical form of the final product and may also influence the release curve of growth factors . A study with 4 subjects, by Pulcini et al.,  describes platelet yield by apheresis, measures select platelet growth factors with respect to their osteogenic potential and identifies the genes activated by the PRP components enhancing the osteogenic potential of cell lines used for in vitro study. The study by Ojea-P´erez et al.,  talks about large scale production (553 samples) of PRP in a closed blood bank like setup. This study aptly describes the method of preparation of frozen PRP with monitoring of the quality/characteristics at regular intervals, showing almost no significant deterioration of quality/characteristics over time. Before using, the frozen PRP is thawed and activated using CaCl2, which will help in release of factors from the platelets. The study supports its utilization in bone healing and regenerative medicine. A study by Ulasliet al., , recruited 12 subjects, talks about collection of whole blood in sodium citrate (SC) 0.3% solution as anticoagulant, which follows 3 different protocols, blood collection in tubes with 0.9ml SC in protocol 1, 0.5ml SC in protocol 2 and no SC in protocol 3. The PRP obtained from protocol 1 had high platelet count, highest TGF-b1 (transforming growth factor b1) and PDGF-BB levels, while protocol 3 had elevated levels of WBC’s in PRP, correlating positively with highest concentration of VEGF (Vascular Endothelial Growth Factor) and total VEGF levels (p<0.001 for both and r:0.693, r:0.603 respectively). Another study by Machado et al. , recruited 32 subjects and used a remarkably simple, economical and highly reproducible approach called Turn Down-Turn Up PRP Protocol Double Spin-Closed System, claiming of results which are consistent with current standards of quality for PRP preparation for clinical trials suggested by the American Academy of Orthopedic Surgeons working group, who presented the MIBO statement. Their PRP preparation in a hospital facility is very economical, without considering the expenses of equipment and laboratory personnel. Another interesting study by Baba et al.,  used Umbilical cord blood derived PRP (UCB-PRP) from 9 healthy deliveries and compared its effect (osteogenic potential) on cryopreserved autologous Mesenchymal stem cells (MSC) derived from respective patients, after 3 years of storage. The study primarily considered secretion of 3 platelet derived factors platelet-derived growth factor-BB (PDGF-BB), an MSC proliferation accelerator, transforming growth factor β1 (TGF-β1) of TGF-β superfamily, an extracellular matrix production accelerator; and vascular endothelial growth factor (VEGF), an angiogenesis accelerator and measured the osteogenic potential of UCB-PRP, which was found to be intact even after 3 years of cryopreservation. The study did not investigate any adverse effects of cryopreservation on PRP or MSC’s. The observed studies had variations in procedures with very few similarities such as platelet yield and all of them measured and characterized GF’s. Even though their numerical data is available, statistical comparisons of these two parameters across different studies may be non-significant/difficult due to differences in their approach to get platelet yield or number and type of GF/platelet derived factors considered.
Patient variability: Demographic variables like age, gender, presence and absence of comorbidities, medication usage must be considered as they address the pathophysiological variations and hence the quality of PRP derived from the patients . The in vitro study by Pulcini et al.,  recruited 4 subjects between the age 21 years and 59 years, none were on anti-platelet medications, and they studied the platelet yield and GFs (Growth Factors) in their samples. They applied the freeze-thaw cycles to activate platelets before using them to stimulate osteogenic growth in artificial human cell line cultures. The study notes variations in cytokines and GFs between aged and young subjects consistent with previous studies . Higher levels of GF’s were noted in females and subjects < 25 years of age. The study acknowledges reports from previous studies where more pro-inflammatory cytokines were noted in the aged population in comparison to young subjects. However, it recommends considering larger sample sizes to draw more conclusions. In consistent with previous studies, higher concentration of cytokines and GFs had no relation to cell proliferation in cell lines used for testing PRP efficacy during in vitro studies [22,23]. This raises an important question, what should be the ideal concentration of platelets, cytokines, and GFs to facilitate cell growth and proliferation in a particular tissue type. A study by Pulcini et al.,  noted the effect of different dilutions of PRP on the proliferation of cells in commercial human cell lines. Another study by Velier et al.,  used PRP/PRP gel in chronic wound care and addresses the patient related variability. The study compared 5 young healthy adults (median age 23 years) to 5 elderly patients (median age 85 years) with comorbidities like acute coronary syndrome, acute renal failure, stroke, obesity, hypertension, and diabetes, and on medications, with additional prescription for aspirin, clopidogrel and fluindione. They found that the intensity expression of P-selectin after ADP stimulation was significantly higher in healthy donors compared to elderly (p = 0.03), whereas the basal expression of P-selectin showed no statistical difference. P-selectin expression is a measure of platelet activation. Prothrombin concentration (p = 0.01) was measured higher in healthy donors. The researchers did not notice any significant difference in the biological characteristics of the therapeutic product PRP gel. The quality of the gel was correlated to the rate of formation. The faster the gel was formed, the more consistent it was. Linear correlations were found between biological parameters and PRP gel time formation. Overall, this study indicates that antithrombotic drugs have no impact on platelets functionality in PRP and PRP gel production. The limitations of the study were the small sample size, lack of evaluation on effects of oral anticoagulant therapy on PRP gel formation, and the PRP processing method had lower platelet yield in comparison to other recorded methods .
Characterization of PRP: The product derived following a standardized preparatory protocol requires desired measurable characteristics. This is an indirect measure of the potential of the preparatory procedure followed. The measurable characteristics will have to be in a range defined by different professional organizations . The measurable characteristics of PRP (In comparison with whole blood/plasma measurements) are platelet count/folds of platelet concentration, levels of GF’s, cytokines, chemokines, proinflammatory and anti-inflammatory factors, adhesion molecules, lysosomal enzymes, extracellular vesicles. The PRP contains a diverse group of factors which collectively are responsible for the therapeutic outcomes . But there are no studies available which consider the broader picture. This is due to the extensive nature of the parameters which need to be considered and the limitation of resources available for the process . Different cell types and tissues have variable requirements of growth factors or cytokines for their proliferation, also multiple of these factors can have overlapping functions in different cell types . This highlights the need to consider the tissue specific requirements of the constituents in PRP /PRP products. Majority of the PRP studies have involved the musculoskeletal system and Aesthetic medicine . Most of the PRP preparation methods considered here can obtain higher platelet concentrations as compared to whole blood. These samples on stimulation or mixing with activators, can release various platelet factors which are the ultimate effectors of PRP therapy. Some studies have appreciated few WBCs in their PRP preparation. These studies claim that presence of certain types and number of WBCs in PRP may help in healing musculoskeletal injuries . The activated PRP shows elevated levels of several factors, the composition of which has been characterized in several in vitro studies using standard ELISA based kits [3,12,14]. No study was found to have described all the constituents of PRP.
In vitro studies. Studies have measured platelet derived factors in PRP and were able to demonstrate specific pro-osteogenic gene activation in MSC derived commercial human cell lines [11,13,17] and umbilical cord derived human autologous MSC cell lines . The product was characterized in terms of platelet yield and growth factors, proliferation of the MSC’s in cell lines, and activation of specific genes was noted to measure the efficacy of the product.
In vivo studies: Study by Popescu et al  used commercially available kits, all requiring blood collection and centrifugation, delivering injectable products which were divided into distinct groups and compared as 1. A-PRP (Activated PRPR) 2. Non activated PRP Vs Calcium activated PRP 3. Human follicle mesenchymal stem cells plus A-PRP. 4. PRP plus Hyaluronic acid (HA) biofunctionalized scaffolding. 5. PRP with fat graft and Adipose derived MSC. Variations were seen in the quality of the product prepared using vacuum blood collection tubes with anticoagulant, anticoagulant prefilled syringes, and blood collection bags with anticoagulant. The product was characterized in terms of platelet yield and growth factors, activation of specific genes was noted, and clinical efficacy was the ultimate measure of the product's quality. Study by Velier et al., demonstrating the PRP’s wound healing potential shows that it can be converted into gel form and applied as dressing. This emphasizes the efficacy of the PRP in treatment of chronic ulcers . Musculoskeletal system-based studies have demonstrated safety and efficacy of PRP in human studies [1,9]. We are now looking at the 4th generation approach where it is possible to design methods (Using engineering techniques) to maintain slow and sustained release of platelet derived factors in vivo [11,13].
When we discuss standardization of PRP preparation procedures, we must consider the commonality in the various reported PRP preparation procedures. The desired outcomes of PRP preparation are 1. high yield of platelets, 2. enhanced level of platelet derived factors in activated PRP, 3. sterility of the product, 4. reproducibility. A 2013 study by Amable et al.  describes a simple and reproducible technique used to produce PRP from 22 different subjects. It reported the highest yield of platelets (8.8 to 9.3 folds after 2nd spin), more than by any study so far. The study uses 5 ml tubes containing 3.2% sodium citrate BD vacutainers with 0.5ml SC anticoagulant, subjected to a double spin method at low speeds (300g X 5 min 1st spin and 700g X 17 min 2nd spin) and variable temperature (12 degree C and 18 degree C). The temperature differences did not lead to any statistically significant platelet yield differences. The platelet pellets obtained after 2nd spin were resuspended and activated using CaCl2 and/or thrombin. The sample was centrifuged at 3000g to measure GF’s and other platelet factors. The GF levels were variable in comparison to other previous studies, but it showed increased alpha granule release by calcium. Anti-inflammatory cytokines and chemokines showed no major differences between plasma and activated PRP. Proinflammatory cytokines are the ones that showed a significant increase in activated PRP. There are similar studies in the recent past claiming to develop high yield PRP. Therefore, we suggest that rather than focusing too much on various procedures for a good yield, irrespective of the procedures, we should focus on achieving a desired range of platelet yield, desired quality of PRP and desired level of platelet derived factors from that yield. In other words, getting a product which is enhanced in all parameters. The final product may be highly concentrated, in terms of constituents and can be diluted to desired proportions for its in vitro or in vivo or tissue specific application. The study by Pulcini et al.  points towards an interesting approach where serial dilutions of PRP can help us determine the optimum level of Platelets and factors customized for a particular tissue or pathological condition. This approach finds support in the evidence found in a study conducted on 29 subjects by Cassano et al  where bone marrow aspirate (BMA) was collected by the same surgeon. Each sample was divided and processed using two types of commercially available kits. PRP was processed using a standardized kit and method. The biologic autologous conditioned serum (ACS) was derived from it and used to test the osteogenic potential of BMA derived stem cells, like in prior studies [24,25]. Factors like IL-1ra, present in ACS, correlated positively with osteogenic activity [16,26]. This study highlights usage of tissue specific requirements parameters to define quality of the PRP. This is in line with other musculoskeletal in vitro studies which have highlighted the effect of specific platelet derived factors on the osteogenic potential of stem cell lines [11,13,17]. A study involving 15 subjects by Bernardi et al.,  also highlights the fact that freeze-thawing technique for PRP can damage platelet membranes and may potentially affect the quality of PRP, especially in terms of slow and sustained release of factors in vivo. This could affect the clinical outcome of PRP. These findings can assist us in selecting between fresh prepared PRP and freeze thawed PRP for specific applications. For in vivo effects, the interaction of injected PRP and/or platelet derived factors with the local tissue depends on the characteristics of the product, and the altered pathophysiological parameters at the site of lesion. Some randomized studies have documented this interaction in osteoarthritis [26,27]. More randomized studies are needed to understand the full scope of the nature of this interaction in different tissues as the requirements can be tissue specific .