|Year : 2021 | Volume
| Issue : 4 | Page : 250-254
Evaluation of factors affecting the quantity and quality of platelet-rich plasma and platelet-derived growth factor-BB
Rahul Verma1, Priyanka Sharma2, Gita Negi1, Abhishek Kandwal3, Harish Chandra1
1 Department of Pathology, Himalayan Institute of Medical Sciences, Dehradun, Uttarakhand, India
2 Department of Pathology, GMC, Jammu, India
3 Department of Dental Surgery, Himalayan Institute of Medical Sciences, Dehradun, Uttarakhand, India
|Date of Submission||05-Dec-2020|
|Date of Acceptance||29-Jul-2021|
|Date of Web Publication||27-May-2022|
SRL LTD, Dr. RPGMC Tanda, Himachal Pradesh
Source of Support: None, Conflict of Interest: None
Context: Platelet-rich plasma (PRP) releases growth factors (GFs) upon activation which accelerates the healing and regeneration in the target tissue. Wider applications of PRP warrant product standardization. PRP compositions may vary according to the patient's demographics and the constituents of PRP. Aims: Our study aimed to assess the correlations between age, gender, platelet counts, platelet indices, platelet yield, platelet dose, GF dose, and platelet-derived GF-BB (PDGF-BB) concentration. Settings and Design: This was an experimental study done in the Department of Pathology along with the Department of Dentistry at a tertiary care hospital in North India. Subjects and Methods: PRP was prepared from 40 ml of whole blood. Platelet counts, platelet indices (LH 750 coulter analyzer, Florida, USA), and PDGF-BB levels (enzyme-linked immunosorbent assay, Qayee Bio., Ltd, China) were measured. Platelet yield (%), platelet dose (×106), and GF dose (×1012) in PRP were also calculated. Statistical Analysis Used: All the parameters were analyzed using Pearson's correlation coefficient and linear regression. Paired t-test and Mann–Whitney test were used for two-group analysis. Results: Mean platelet count, PDGF-BB concentration, platelet yield, platelet dose, and GF dose in PRP were 1317 × 103/mm3, 30 ± 9.89 ng/ml, 71.62 ± 28.34%, 6.5 ± 3.5 × 109, and 159.62 ± 52.39 ng/ml, respectively. PRP platelet counts were positively correlated with PDGF-BB concentration, platelet yield, platelet dose, and GF dose. Conclusions: Of all the parameters, PDGF-BB concentration, platelet yield, platelet dose, and GF dose showed a positive correlation with PRP platelet count. Age, gender, and platelet indices had no significant influence.
Keywords: Growth factor dose, platelet-derived growth factor-BB, platelet dose, platelet indices, platelet yield, platelet-rich plasma
|How to cite this article:|
Verma R, Sharma P, Negi G, Kandwal A, Chandra H. Evaluation of factors affecting the quantity and quality of platelet-rich plasma and platelet-derived growth factor-BB. CHRISMED J Health Res 2021;8:250-4
|How to cite this URL:|
Verma R, Sharma P, Negi G, Kandwal A, Chandra H. Evaluation of factors affecting the quantity and quality of platelet-rich plasma and platelet-derived growth factor-BB. CHRISMED J Health Res [serial online] 2021 [cited 2022 Jul 6];8:250-4. Available from: https://www.cjhr.org/text.asp?2021/8/4/250/346098
| Introduction|| |
Preparations of platelets generically referred to as platelet-rich plasma (PRP) have gained popularity over the last decade in many fields of medicine, primarily in orthodontics, orthopedics, surgery, and cosmetology. The autologous nature of PRP, its ease of application, and relatively low cost are some advantages of PRP that has led to research interest and a wide clinical application. The term PRP can be defined as the volume fraction of blood plasma, which has 3–5 folds increased concentration of platelets, from a baseline serum level. PRP enhances the healing process by stimulation revascularization and regeneration, owing to the increased concentration of the growth factors (GFs) present inside the alpha granules. It predominantly contains an increased concentration of platelet-derived GF (PDGF), responsible for chemotaxis, cell differentiation, angiogenesis, and osteoblastic proliferation. PDGF-AA, PDGF-AB, and platelet-derived GF-BB (PDGF-BB) are the three major isoform which are released upon platelet activation. Inconsistency in the platelet concentration of the PRP may lead to the variation in the GF concentration vital to attain desired regenerative and healing effect in the tissue. Several quality assessment variables contribute to the inconsistency in the composition of PRP, which ultimately affects the GF concentration. These are the age and gender of the patients, preparation method, PRP platelet count, and platelet indices such as platelet distribution width (PDW) and mean platelet volume (MPV). This study aimed to assess the PDGF-BB levels and to assess the correlation between demographic factors, platelet indices, and platelet counts in PRP.
| Subjects and Methods|| |
This experimental study was carried out in the Department of Pathology along with the Department of Dentistry at a tertiary care center in north India. Whole blood was drawn from 35 cases who were subjected to various dental regenerative procedures after obtaining written informed consent and getting approval from the ethical committee. Subjects having platelet count <150,000/mm3, hemoglobin <10 g/dl, recent fever or illness or septicemia were excluded.
PRP was prepared from 40 ml whole blood collected in acid citrate dextrose-acetate (ACD-A) vials using two spin technique (1600 rpm for 15 min and 2800 rpm for 7 min). After the first spin supernatant containing platelets as well as plasma was aspirated from the ACD-A vacutainers using a 20 G spinal needle, without disturbing the buffy coat and transferred to a conical tube and centrifuged at 2800 rpm for 7 min. Platelet pellet (PRP) at the base of the conical tube along with 5 ml of plasma was left for 1 h at room temperature without disturbing the sediments which were resuspended and kept it an agitator for another 2 h.
Platelet counts and platelet indices (MPV and PDW) of whole blood samples and PRP were determined using automated cell count analyzer (Coulter LH 750, Beckman Coulter, Miami, FL, USA). Platelet yield in PRP (%), platelet dose in the PRP (×109), and GF dose (×1012) were calculated using the following formulas. PDGF-BB levels were measured using enzyme-linked immunosorbent assay kit by Qayee Bio Ltd, China.
Statistical analysis was done using R software (v 3.6.0) (Auckland, Newzeland). All the parameters were analyzed using Pearson's correlation coefficient. Paired t-test was used to analyze platelet counts and platelet indices in whole blood and PRP. Linear regression analysis was performed to evaluate the association between PDGF-BB and PRP platelet counts. Mann–Whitney test was used for gender-based two group comparison for all parameters. A statistical significance level of P < 0.05 was set in all tests.
Results: A total of 35 cases were included in this study with a mean age of 32 years (±9.01). The maximum number of cases were in the age group of 26–35 years (n = 19, 48.7%). Out of 35 cases, 62.8% (n = 22) were females and 37.2% (n = 13) were males. Whole blood platelet count varied from 150 × 103/mm3 to 453 × 103/mm3 with the maximum number of cases (71.4%, n = 25/35) having platelet count within the range of 150–250 × 103/mm3. Platelet count in PRP varied from 425 × 103/mm3 to 2876 × 103/mm3 with the mean concentration achieved as 1317 × 103/mm3. The maximum number of cases 15/35 (42.8%) had platelet count within the range of 800–1200 × 103/mm3. PRP concentration of 5–10 times was achieved in the maximum number of cases (n = 19, 54.2%). Mean platelet yield, platelet dose, and GF dose were 71.62 ± 28.34%, 6.5 ± 3.5 × 109, and 159.62 ± 52.39 ng/ml, respectively.
Platelet counts, MPV, and PDW of whole blood and PRP were significantly different with a P value of 0.001, 0.04, and 0.01, respectively (paired Z test, significance level: 0.05). [Table 1] shows the correlation analysis of all the variables. A positive correlation was found between whole blood platelet counts and platelet counts in PRP. Furthermore, PRP platelet counts were positively correlated with PDGF-BB concentration, platelet yield, platelet dose, and GF dose. PDGF-BB concentration in PRP varied from 22 ng/ml to 71 ng/ml with mean concentration of 30 ± 9.89 ng/ml. Linear regression analysis between PDGF-BB and PRP platelet count indicated a strong relationship with a very high adjusted R2 value of 0.80 [Figure 1]. None of the variables except the PDW of PRP was significantly different between males and females [Table 2].
|Figure 1: Correlation between platelet-rich plasma platelet count and platelet-derived growth factor in platelet-rich plasma|
Click here to view
| Discussion|| |
PRP has been used extensively in various clinical settings over the past decade. However, there are inconsistencies in the results since no clear-cut set of parameters is available for the PRP preparation neither for manual preparation nor for the automatic devices. This causes variations in the PRP concentrations which further leads to variability in the PDGFs. The platelet concentration of the PRP preparation is crucial however merely concentrating the platelets highly does not indicate a quality PRP product. PRP platelet concentration in our study ranged from 2.7 to 13.3 with a mean concentration level of 5.79 times the baseline counts. Various authors have reported that over concentrating the platelets does not enhance the regenerative properties of PRP and that the ideal concentration should be between 2 and 10 folds the baseline platelet count. These findings were supported by the fact that high concentrations of platelets having increased GF concentrations are more likely to over-saturate the limited GF receptors, causing increased scar tissue formation and potential delay in healing.
Although platelet indices (MPV and PDW) showed significant difference with P < 0.05, they were within the physiological reference range. This signifies that neither the anticoagulant nor the method of preparation leads to preactivation of the PRP sample before application at the surgical site. Evaluation of these parameters may serve as a quality control measure for the PRP activation.
No significant association was found between the age and other parameters. Many authors suggested that age and gender did not influence the platelet concentration or on the concentration of GFs. In contrast, some authors found that platelet yield and GF concentration increase with age. [Table 3] shows a comparison between various studies which analyzed age with other variables.
|Table 3: Review of studies showing correlation of age as a function with other variables|
Click here to view
Similarly, most of the variables showed no significant difference between males and females except the PDW of PRP. In contrast to our study, some authors suggest that there exists an association between gender and GF concentration with males having higher concentration as compared to females. However, the reason for gender-based differences could be multifactorial and still needs to be evaluated on a larger cohort with gender and age-specific distributions.
PGDF-BB concentration and PRP platelet count showed a strong linear correlation with a very high adjusted R2 value of 0.80. Many authors had reported similar results in the past. In contrast to our study, Harrison et al. stated that the correlation may not be linear for every individual.
Platelet yield is also one of the quality parameters which influences the regenerative potential of the PRP as well as the clinical outcome. Geetha et al. found a positive correlation of platelet yield with age (r = 0.38) and predonation platelet count (r = 0.42). In contrast, our study did not show any significant correlation of platelet yield with age and whole blood (r = 0.089 and r = 0.088, respectively). Platelet yield being derived from the platelet counts of PRP will always show a positive correlation with it (r = 0.80).
Platelet dose can assess the quality of the PRP and the clinical implications of variability in the PRP products. Magalon et al. gave DEPA classification (Dose of injected platelets, Efficiency of production, Purity of the PRP, Activation of the PRP) to standardize the PRP composition. They categorized the platelet dose as A, very high dose of injected platelets of >5 billion; B, high dose of injected platelets, from 3 to 5 billion; C, medium dose of injected platelets, from 1 to 3 billion and D, a low dose of injected platelets, <1 billion. However, they also stated that the platelet dose depends upon the PRP volume and the PRP preparation protocol. We achieved very high mean platelet dose of 6.5 billion which was significantly higher when compared to the manual protocols used in previous studies.,,,,, Magalon et al. and Castillo et al. found a positive correlation between platelet dose and GF dose (r = 0.40; P < 0.01 and r = 0.43; P = 0.05, respectively). Our results were in concordance with these studies (r = 0.74; P < 0.0001).,,
Our study was limited because small sample size, host modulation factors, and lifestyle of each donor are going to affect the outcomes of the study. Apart from PDW and MPV factors such as pH of the PRP, platelet swirling and leukocyte count were not estimated. These factors are also vital in the quality assessment of the PRP and influence the outcome of the product. We suggest that further studies should be undertaken to evaluate the above-mentioned parameters with a control group for the product standardization. Furthermore, it is pertinent to mention that the effect of different anticoagulants like CPDA (citrate phosphate dextrose adenine) on the PRP concentration was not compared with ACD. Quantification of other GFs was beyond the scope of this study. Further research is mandated considering these variables to understand the healing and regenerative effects of PRP as well as to limit the final product variability which will further help in the standardization of the protocol for PRP preparation.
In our study, age and gender did not show any significant influence on other parameters. PDGF-BB concentration, whole blood, and PRP platelet counts showed a positive correlation with each other. Moreover, PDGF-BB concentration and PRP platelet counts showed a strong linear correlation. Platelet yield, platelet dose, and GF dose also showed a positive correlation with PRP platelet count.
Quality, as well as the quantity of the PRP product, needs to be standardized with specific protocols considering individual parameters as mentioned in our study. Further inclusions of more variables in a larger cohort are required to achieve better clinical efficacy of the PRP products.
Financial support and sponsorship
This study was financially supported by Himalayan Institute of Medical Sciences.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Boswell SG, Cole BJ, Sundman EA, Karas V, Fortier LA. Platelet-rich plasma: A milieu of bioactive factors. Arthroscopy 2012;28:429-39.
Weibrich G, Kleis WK, Hafner G, Hitzler WE. Growth factor levels in platelet-rich plasma and correlations with donor age, sex, and platelet count. J Craniomaxillofac Surg 2002;30:97-102.
Colciago A, Celotti F, Casati L, Giancola R, Castano SM, Antonini G, et al. In vitro
effects of PDGF isoforms (AA, BB, AB and CC) on migration and proliferation of SaOS-2 osteoblasts and on migration of human osteoblasts. Int J Biomed Sci 2009;5:380-9.
Bowen-Pope DF, Vogel A, Ross R. Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide spectrum of agents. Proc Natl Acad Sci U S A 1984;81:2396-400.
Textor J. Platelet-Rich Plasma (PRP) as a therapeutic agent: Platelet biology, growth factors and a review of the literature. In: Lana JF, Andrade Santana MH, Dias Belangero W, Malheiros Luzo AC, editors. Platelet-Rich Plasma. Lecture Notes in Bioengineering. Berlin Heidelberg: Springer; 2014. p. 61-94.
Tynngård N. Preparation, storage and quality control of platelet concentrates. Transfus Apher Sci 2009;41:97-104.
Arora S, Agnihotri N. Platelet derived biomaterials for therapeutic use: Review of technical aspects. Indian J Hematol Blood Transfus 2017;33:159-67.
Bae JH, Kim YK, Myung SK. Effects of platelet-rich plasma on sinus bone graft: Meta-analysis. J Periodontol 2011;82:660-7.
Plachokova AS, Nikolidakis D, Mulder J, Jansen JA, Creugers NH. Effect of platelet-rich plasma on bone regeneration in dentistry: A systematic review. Clin Oral Implants Res 2008;19:539-45.
Sheth U, Simunovic N, Klein G, Fu F, Einhorn TA, Schemitsch E, et al.
Efficacy of autologous platelet-rich plasma use for orthopaedic indications: A meta-analysis. J Bone Joint Surg Am 2012;94:298-307.
Yamaguchi R, Terashima H, Yoneyama S, Tadano S, Ohkohchi N. Effects of platelet-rich plasma on intestinal anastomotic healing in rats: PRP concentration is a key factor. J Surg Res 2012;173:258-66.
Krasna M, Domanović D, Tomsic A, Svajger U, Jeras M. Platelet gel stimulates proliferation of human dermal fibroblasts in vitro
. Acta Dermatovenerol Alp Pannonica Adriat 2007;16:105-10.
Everts PA, Hoffmann J, Weibrich G, Mahoney CB, Schönberger JP, van Zundert A, et al.
Differences in platelet growth factor release and leucocyte kinetics during autologous platelet gel formation. Transfus Med 2006;16:363-8.
DeLong JM, Beitzel K, Mazzocca AD, Shepard D, Roller BL, Hanypsiak BT. Update on platelet-rich plasma. Curr Orthop Pract 2011;22:514-23.
Giovanetti TV, do Nascimento AJ, de Paula JP. Platelet indices: Laboratory and clinical applications. Rev Bras Hematol Hemoter 2011;33:164-5.
Taniguchi Y, Yoshioka T, Sugaya H, Gosho M, Aoto K, Kanamori A, et al.
Growth factor levels in leukocyte-poor platelet-rich plasma and correlations with donor age, gender, and platelets in the Japanese population. J Exp Orthop 2019;6:4.
Cho HS, Song IH, Park SY, Sung MC, Ahn MW, Song KE. Individual variation in growth factor concentrations in platelet-rich plasma and its influence on human mesenchymal stem cells. Korean J Lab Med 2011;31:212-8.
Dragoo J, Korotkova T, Wasterlain A, Pouliot M, Kim H, Golish S. Age-related changes of chondrogenic growth factors in platelet-rich plasma. Oper Techniq Orthop 2012;22:49-55.
Arun R, Yashovardhan A, Deepthi K, Suresh B, Sreedhar Babu K, Jothibai D. Donor demographic and laboratory predictors of single donor platelet yield. J Clin Sci Res 2013;2:211-5. [Full text]
Geetha CP, Korti P, Jayashankar E, Deshpande A. Factors affecting platelet yield in single donor plateletpheresis: A single institution experience. Indian J Pathol Oncol 2017;4:23-6.
Evanson JR, Guyton MK, Oliver DL, Hire JM, Topolski RL, Zumbrun SD, et al.
Gender and age differences in growth factor concentrations from platelet-rich plasma in adults. Mil Med 2014;179:799-805.
Xiong G, Lingampalli N, Koltsov JC, Leung LL, Bhutani N, Robinson WH, et al.
Men and women differ in the biochemical composition of platelet-rich plasma. Am J Sports Med 2018;46:409-19.
Lee JW, Kwon OH, Kim TK, Cho YK, Choi KY, Chung HY, et al.
Platelet-rich plasma: Quantitative assessment of growth factor levels and comparative analysis of activated and inactivated groups. Arch Plast Surg 2013;40:530-5.
Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation. J Oral Maxillofac Surg 2000;58:297-300.
Harrison S, Vavken P, Kevy S, Jacobson M, Zurakowski D, Murray MM. Platelet activation by collagen provides sustained release of anabolic cytokines. Am J Sports Med 2011;39:729-34.
Singh RP, Marwaha N, Malhotra P, Dash S. Quality assessment of platelet concentrates prepared by platelet rich plasma-platelet concentrate, buffy coat poor-platelet concentrate (BC-PC) and apheresis-PC methods. Asian J Transfus Sci 2009;3:86-94.
] [Full text]
Magalon J, Chateau AL, Bertrand B, Louis ML, Silvestre A, Giraudo L, et al.
DEPA classification: A proposal for standardising PRP use and a retrospective application of available devices. BMJ Open Sport Exerc Med 2016;2:e000060.
Magalon J, Bausset O, Serratrice N, Giraudo L, Aboudou H, Veran J, et al.
Characterization and comparison of 5 platelet-rich plasma preparations in a single-donor model. Arthroscopy 2014;30:629-38.
Castillo TN, Pouliot MA, Kim HJ, Dragoo JL. Comparison of growth factor and platelet concentration from commercial platelet-rich plasma separation systems. Am J Sports Med 2011;39:266-71.
[Table 1], [Table 2], [Table 3]