How Rich is Platelet Rich Plasma? A Simple Preparation Method of PRP and Its Quality According to Various Classification Systems

Background: platelets are a rich source of various growth factors and hence platelet rich plasma (PRP) is being used therapeutically in the ﬁ eld of dermatology, orthopaedics and dentistry with promising results. However, methods of preparation of PRP vary with many commercial kits also available. Scoring systems like DEPA score, PAW system and sports medicine criteria score help in determining the quality of the ﬁ nal product and guide therapy. Here we test a simple two step method for preparation of PRP and score it according to the abovementioned criteria while comparing it with the commercial kits analysed in literature. Methods: 10 ml whole blood was collected in 1.5 ml ACD solution from 100 healthy willing participants and analysed for platelet concentration. Sample was then centrifuged at 1600 rpm X 4 minutes in a calibrated laboratory centrifuge and supernatant was collected in a sterile tube using a sterile syringe. The supernatant was further concentrated for platelets by centrifuging at 3600 rpm X 10 minutes to form a platelet pellet at the bottom. All but 1 ml supernatant (Platelet de ﬁ cient plasma) was discarded and the platelet pellet was suspended again to form platelet rich plasma. Platelet count was estimated in this sample as done with the whole blood sample. The results were compared and analysed using statistical methods to determine the ef ﬁ cacy of concentrating platelets by this method. Results: The tested method gave a ﬁ nal concentration of more than four times the baseline concentration with approximately 30% ef ﬁ ciency of capture and 80% purity. This was comparable to majority of the commercial systems tested in literature at a fraction of the cost. The scores according to various classi ﬁ cation systems were determined and reported. Conclusion: Every platelet rich plasma prepared for therapeutic purposes should be classi ﬁ ed according to any of the available scoring systems to determine its suitability for the purpose and to maintain uniformity in the quality of the product. The proposed two step method of preparation yields satisfactory quality of PRP by utilizing services of a basic laboratory at a fraction of the cost of commercial systems thereby enabling smaller medical institutions to utilize PRP therapy.

stimulating hair growth in cases of androgenic alopecia, hastening bone formation in cases of autologous bone grafting etc.
Studies have proposed diff erent characterisation criteria for PRP like the DEPA classifi cation which takes the following parameters into account: (1) Dose of injected platelets, (2) Effi ciency of production, (3) Purity of PRP and (4) Activation process (Table 1). Other classifi cation methods including PAW system of classifi cation ( Figure 1) and the sports medicine criteria take in to account (1) WBC concentration, (2) activation and (3) fi nal concentration ( Table 2) 15,16 .

THE PARAMETERS OF PRP
Th e diff erent parameters used in these classifi cation systems need to be understood to correctly choose a platelet rich plasma preparation for each specifi c intended purpose.
A. Platelet concentration/ Dose of injected platelets: the quantifi cation of platelets in PRP can be done either in terms of times increase of baseline count or as an absolute platelet count per microlitre. Th e best results have been found with platelet concentrations ranging from 2 times to 6 times the baseline platelet concentration corresponding with absolute values greater than baseline and going up to 1800000/uL [17][18][19] . Th e
Recently, commercially available kits have sprung up which guarantee a higher yield of platelets from a small amount of blood but incur a high cost of production 14 .
To circumvent the cost and still reap the probable benefi ts of this experimental treatment, many practitioners resort to simply centrifuging small amounts of blood without following specifi c guidelines and use the supernatant so obtained thereby leading to lower yield of platelets, higher amount of dosage in terms of volume, greater degree of pain and ultimately poor patient acceptance and results.
As the use of PRP is growing exponentially because of the publicity and the easy availability, there needs to be a proper guideline for harvesting platelets from a small amount of blood and to classify the end product in a universal score to determine its quality before being used for a variety of therapeutic purposes including D. Activation of PRP: Various exogenous methods like calcium chloride, thrombin and type 1 collagen can be added to activate platelets in vitro before use. Whether exogenous activation leads to better results or not needs to be investigated further but adverse eff ects such as lowering of pH of PRP and subsequent increase in pain on addition of calcium chloride need to be considered.

MATERIAL AND METHODS
Time period: July 2019 to Oct 2019 Study setting: Defence services teaching hospital Sample size: 100 Inclusion criteria: a) Samples from consenting healthy individuals who reported for routine medical examination. b) Samples which did not show presence of platelet clumps on analysis Exclusion criteria: a) Samples from individuals who have reported to the hospital for some illness. b) Samples which showed presence of platelet clumps on analysis during any step. Procedure a) 10 ml blood was collected in 1.5 ml ACD solution in sterile tubes and mixed using inversion technique. b) Whole blood sample was analysed for platelet concentration using Beckman Coulter haematology analyser and results noted.
dose of injected platelets can be determined by multiplying the fi nal volume used by the concentration. However, it should be borne in mind that higher concentrations don't always mean better results as studies have shown inhibitory eff ect on growth at concentrations having greater than 6-fold increase in the fi nal product. Th e underlying mechanism may be a paradoxical inhibition due to alteration in the healing milieu by downregulation of growth factor receptors, receptor desensitisation and stimulation of apoptosis 19 . B. Effi ciency of capture: the effi ciency of capture of any method can be calculated by determining the percentage of total platelets from the whole blood sample that have been captured in the PRP. A majority of the commercially available kits analysed by Magalon et al had an effi ciency rate ranging from 30 to 70%15. C. Purity of preparation/ WBC concentration: Purity of preparation signifi es the relative composition of platelets, leucocytes and RBCs in the obtained PRP with a relative concentration of platelets above 90% being classifi ed as very pure PRP. Th e desirability of WBCs in the fi nal product has been under scrutiny and depends on the intended use of the PRP. Th erefore, it should be assessed and reported while classifying the end product as either below baseline or above baseline WBC levels.  WBC count: lesser than baseline count in 97% of samples. Activation: not activated exogenously.
Th erefore, the average fi nal score according to the three diff erent classifi cation systems discussed above would be as follows 1. DEPA Score: if 1 ml of PRP made from 10 ml whole blood: DCB 2. PAW Score: P3-B 3. Sports medicine criteria score: Type 4B

DISCUSSION
Th e quality of PRP prepared by the abovementioned method was evaluated using the available scoring systems in literature and compared with other studies.
DEPA score: Th e fi nal DEPA score of our preparation was DCB. Magalon et al did a thorough search of literature and recorded the DEPA scores of PRP obtained from commercial kits and homemade methods used in various studies including their own15.
Th e dosage of injected platelets was 0.9 billion platelets if 1 ml of PRP was prepared from 10 ml of blood which characterized it as "D" as per Table 1. Out of the eleven methods which were tested by various researchers and which used similar amount of whole blood to harvest platelets, ten had the same score. Nine other c) Sample was then centrifuged at 1600 rpm X 4 minutes in a calibrated laboratory centrifuge and supernatant was collected in a sterile tube using a sterile syringe d) Th e supernatant was further concentrated for platelets by centrifuging at 3600 rpm X 10 minutes to form a platelet pellet at the bottom. All but 1 ml of the supernatant (Platelet defi cient plasma) was discarded and the platelet pellet was resuspended to form 1 ml of platelet rich plasma. e) Platelet count was estimated in this sample as done with the whole blood sample. f ) Th e results were compared and analysed using statistical methods to determine the effi cacy of concentrating platelets by this method

RESULTS
PRP prepared from 100 samples by the proposed two step method was evaluated for quality and the following were observed (Figure 2).

CONCLUSION
Every PRP should be scored according to any of the criteria which suits the purpose of the institution to determine its quality and suitability for the indication. Th is study illustrates a simple method to harvest a good quality of PRP using the services of a basic laboratory which can prove to be economical and benefi cial in terms of patient outcomes in future clinical studies. Th e following must be kept in mind while evaluating the fi nal concentration of the platelets 1. Th e fi nal concentration depends on the baseline platelet count and hydration status of the individual with a higher baseline count and a well hydrated donor yielding a higher fi nal concentration. 2. Th e centrifugal force applied on the sample may vary between diff erent centrifuges depending on the rotor size of the centrifuge.it is imperative to adjust the other two variables i.e. speed and duration of spin to achieve optimal centrifugal force. Th e centrifugal force in our method for the specifi c instrument used was 430 g x 4 minutes followed by 2200 g x 10 minutes. 3. Platelet count should not be determined immediately after suspending the platelet pellet as the presence of clumps would give erroneous readings.it is recommended to allow the PRP to be placed on a sample roller for 20 minutes before analysis. 4. Th e dose of injected platelets may be increased by drawing more blood and following the same procedure to harvest a higher number of platelets.
Compliance with ethics requirements: Th e authors declare no confl ict of interest regarding this article. Th e authors declare that all the procedures and experiments of this study respect the ethical standards in the Helsinki Declaration of 1975, as revised in 2008 (5), as well as the national law. Informed consent was obtained from all the patients included in the study.
methods which used signifi cantly higher amounts of blood generated a higher dosage of platelets which can be matched by the proposed method by increasing the initial volume. Around 40% of the platelets from the initial sample were captured in the PRP, thereby giving us an efficiency score of "C". Th irteen of the twenty methods tested in literature fared equally at "C" with 4 having lower ("D") and 2 having better ("B") effi ciency. Th is shows comparable effi ciency of the proposed method with commercially available kits.
Purity was determined by relative concentration of platelets among all cellular components of the PRP and ranged from 69% to 91% with an average of slightly above 80%. Th us, scored as "B" which was similar to six and better than ten out of the twenty methods analysed by Magalon et al., with only 4 being better in terms of purity15.
Th e fi nal score as per other classifi cation systems are P3-B (PAW classifi cation) and type 4B (sports medicine criteria) thereby implying a suffi ciently high concentration of platelets with lower count of WBCs Th e DEPA system of classifi cation seems to be better while comparing diff erent methods of preparations as it takes into account contamination also by RBCs in addition to WBCs and has a provision for accounting for absolute effi ciency of capture. Also, the dosage of platelets can be increased by drawing larger amounts of blood to create higher volume of PRP whereas in the other classifi cation systems the platelet concentrations cannot be improved unless higher amount of blood is used to make same amount of PRP.
Th e other two systems for classifi cation provide adequate information about the fi nal PRP to guide therapy as per indication. Th e PRP produced by the proposed method had a score of P3-B in the PAW system and a Sports medicine criteria Type 4B. if exogenously activated the PAW score would be expressed as P3x B.