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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 9  |  Issue : 1  |  Page : 51-53

Comparative evaluation of platelet count in whole blood and injectable platelet-rich fibrin


1 Department of Periodontology, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India
2 Department of General Pathology, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belagavi, Karnataka, India

Date of Submission12-May-2020
Date of Acceptance21-Jan-2021
Date of Web Publication14-May-2021

Correspondence Address:
Sachita Shrikant Naik
Department of Periodontology, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belagavi, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/dmr.dmr_22_20

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  Abstract 


Background: Platelets are formed from megakaryocytes and platelets are present in the circulation for 5–7 days. Platelets are essential in hemostasis, vascular integrity, angiogenesis, inflammation, innate immunity, and wound healing. To capitalize on the advantageous qualities of platelets, platelet-rich fibrin (PRF) was developed by centrifuging peripheral blood. A new platelet concentrate has recently been developed, by utilizing lower centrifugation speed. This new platelet concentrate is in an injectable form called injectable PRF (I-PRF). Aim: This study intends to quantify and compare the platelets in whole blood (WB) and I-PRF. Materials and Methods: The study included ten systemically healthy individuals. Seven milliliter of blood was collected by following standard aseptic protocol. Smears of blood and I-PRF were made and stained by Leishman's stain to confirm the presence of platelets. I-PRF was made by centrifuging 5 ml of blood at 1000 rpm for 3 min at 60 g, which was fed to the automated cell counter unit to count the number of platelets. Results: The platelet count in I-PRF was significantly more when compared with that of WB. Conclusion: The results of our study suggest that the I-PRF has a richer concentration of platelets when compared to the WB.

Keywords: Platelet-rich fibrin, platelets, whole blood


How to cite this article:
Nayak A, Naik SS, Chhatre A, Bhatt A, Paradkar S. Comparative evaluation of platelet count in whole blood and injectable platelet-rich fibrin. Dent Med Res 2021;9:51-3

How to cite this URL:
Nayak A, Naik SS, Chhatre A, Bhatt A, Paradkar S. Comparative evaluation of platelet count in whole blood and injectable platelet-rich fibrin. Dent Med Res [serial online] 2021 [cited 2021 Dec 2];9:51-3. Available from: https://www.dmrjournal.org/text.asp?2021/9/1/51/315962




  Introduction Top


Platelets are small, anucleated, granule containing discoid structures of the terminal stage development of the megakaryocytic series.[1] Platelets play a critical role not only in hemostasis but also in the wound healing process. They are a reservoir of proteins and some growth factors such as platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and insulin-like growth factor (IGF-I) which are required for wound healing.[2] Making use of the natural process of aggregation of activated platelets during hemostasis, an autologous biomaterial was formulated by centrifuging venous blood without any anticoagulant which came to be known as platelet-rich fibrin (PRF).[3] Further research was done to enhance the properties of PRF by changing either the armamentarium used or the methodology, which lead to the discovery of varied formulations of new and different forms of PRF, namely leukocyte-PRF,[4] titanium-prepared PRF,[5] advanced PRF,[6] and the injectable PRF (I-PRF).[7] All the above mentioned forms of PRF have similar components like white blood cells, growth factors, complex 3-dimensional fibrin meshwork, but they vary in the concentration of individual components.[4] I-PRF became a clinical asset in places where PRF was used. In an injectable form, it could be used with various biomaterials to enhance the regenerative property.[7]

To capitalize on the above-said properties of I-PRF, it is essential to confirm that there exists a concentration of platelets in the I-PRF, more so, than that is evident in whole blood (WB). To be able to corroborate this, we decided to undertake this study to quantify the amount of platelets in WB and in I-PRF and compare the two.


  Materials and Methods Top


Ten systemically healthy individuals, aged between 20 and 55 years, were selected from patients visiting the department of periodontology in the institute. The study was conducted after obtaining ethical clearance from the institutional ethical and review board (ethical certificate no. 52, dated: April 13, 2018). Patients with any kind of bleeding/clotting disorders, history of anticoagulants or any other medication, diabetes, hypertension, and malignancy were excluded from the study. Written informed consent was obtained from the patients. Employing proper aseptic procedure, a venous blood sample of 7 ml was collected from the antecubital vein. One milliliter of blood was dispensed immediately in ethylenediaminetetraacetic acid (EDTA) coated tube, shaken well, and fed to an automated cell counter for WB platelet count. One drop of blood was smeared on a clean dry glass slide, stained by Leishman's stain, and viewed in ×40 magnification by a light microscope for the presence of the platelets in the WB. The remaining 5 ml of blood was centrifuged at 1000 rpm, at 60 G for 3 min. The supernatant fluid was collected immediately with the help of a needle syringe. Of this, 1 ml I-PRF was transferred to an EDTA coated tube, shaken well, and fed to the automated cell counter for determination of the platelet count. A drop of I-PRF was also smeared on a clean dry glass slide and stained by Leishman's, stain and the presence of platelets in the I-PRF was viewed in ×40 magnification by a light microscope. An automated cell counter printed a result sheet which mentioned the platelet count of the WB and similarly the platelet count of I-PRF, which was then tabulated.

Statistical analysis

The normality of distribution was tested by the Kolmogorov–Smirnov test, after which the paired t-test was applied.


  Results Top


Kolmogorov–Smirnov test analysis of the data collected indicated that the groups had a normal distribution, The standard deviation of whole blood (WB) (Z value 0.634) and I-PRF (Z value 0.4510) signifies the normal distribution of the samples around the mean in a normal distribution curve. [Table 1].[8]
Table 1: Normality of platelet count in whole blood and injectable platelet-rich fibrin by Kolmogorov-Smirnov test

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A comparison was done between the platelet counts of WB and I-PRF. Paired–t-test was applied to the groups to find the significance of difference. The mean value for WB was 264.50 × 103/ul with a standard deviation of 50.42 and the mean value for I-PRF was 641.50 × 103/ul with a standard deviation of 163.01, implying that there is a statistically significant difference between the sample means. The platelet count was more in the I-PRF when compared to the WB which is evident based on the t value that stands at −8.9923. The value of P 0.0001 implies that a statistically significant difference is seen between the platelet counts of WB and I-PRF [Table 2].
Table 2: Comparison of whole blood and injectable platelet-rich fibrin samples with platelet counts by paired t-test

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When the platelet counts of WB and I-PRF were correlated, a positive linear correlation was observed, as is evident in the scatter plot [Figure 1]. It denotes a significant relationship between the WB and I-PRF where the value of r is 0.7019 implies a moderately positive correlation (as r value lies between 0 and + 1).[9] A statistically significant increase in platelet count of the I-PRF was seen when compared to the platelet count of the WB (r= −0.7019).
Figure 1: Correlation of platelet counts between whole blood and injectable platelet-rich fibrin samples

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  Discussion Top


Miron et al. in 2017 presented I-PRF which is in the injectable form, as one of the recent modifications of PRF.[10] The protocol followed was that of centrifugation of venous blood at a very low speed of 700 rpm at 60 g for a short centrifugation time of 3 min.[10] We conducted a pilot study with two different protocols. In the first protocol, we centrifuged venous blood at 700 rpm with 60 g for 3 min, and in the second protocol, we altered the rpm and centrifuged venous blood at 1000 rpm at 60 g for 3 min. We obtained more number of platelets with the second protocol. Hence, we standardized our study by centrifuging venous blood at 1000 rpm at 60 g for 3 min. Our study results indicated that the platelet count in IPRF was double the platelet count of the WB. Studies done by different authors with various protocols inferred similar results like those of our study, although most used 700 rpm. Wend et al. 2017 followed three different protocols to obtain maximum number of cells in the I-PRF.[11] The protocol which fetched maximum cells was 700 rpm at 60 g for 3 min.[11] Karde et al. in 2018 followed a protocol of centrifugation at 700 rpm for 3–4 min but used 2 ml of venous blood. Platelet count was statistically significant when compared with the other platelet concentrates such as PRF and platetet-rich plasma.[12]

Cellular response to altering the speed of centrifuging might be sensitive to cell-specific properties, such as weight, size, and density of the cells.[11] Choukroun and Ghanaati in 2017 studied these influencing factors, as well as the effect of time variation in the entrapment of the specific cells like platelets in the fibrin meshwork.[13] Similarly, Aggarwal and Singhal 2015 altered the time of centrifugation to obtain a maximum amount of platelets in PRF.[14] Wend et al. 2017 altered the G force from 60 g to 966 g where 60 g delivered a good platelet count.[11] We too standardized the G force at 60 g in our study.

Evaluation of platelet counts can be done by various devices. We used the automated method. A study done by Webb et al.[15] to estimate the platelet count by peripheral smear method and by automated cell counter concluded that there was no statistically significant difference in both the methods.[15] Wend et al. 2017 used an automated device to count the platelets,[11] but Karde et al. 2018 used the smear method as they attributed it to a cost-effective and reliable method.[12]

Platelets release growth factors such as PDGF, TGF-β, and IGF-I once activated and thus play a crucial role in wound healing.[2] To derive the benefits of the rich platelet concentrate of I-PRF, with respect to its antibacterial property,[12] wound healing,[16] and regeneration,[17] its use in a clinical setup is justified.


  Conclusion Top


PRF has seen its place in wound healing, antibacterial property, and regeneration in clinical practice. All the above-said phenomenons are possible due to their rich constitution of platelets, cytokines, and growth factors. Similarly, I-PRF which is a new form of PRF is slowly gaining popularity over PRF. Our study results indicate that I-PRF has double the number of platelets than those seen in PRF. This is worth studying further clinically, such that evidence builds up about its advantages over PRF. It is also noteworthy that I-PRF due to its injectable form may find more applications. Having said this, we hope to inspire more studies to reiterate our findings on the edge that I-PRF has over PRF.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Firkin F, Chesterman C, Penington D, Rush B. Formation of blood cell, bone marrow biopsy. In: Firkin F, editor. De Gruchy's Clinical Haematology in Medical Practice. 5th ed. New Delhi: Wiley India Private Limited Publishing; 2008. p. 10.  Back to cited text no. 1
    
2.
Naik B, Karunakar P, Jayadev M, Marshal VR. Role of platelet rich fibrin in wound healing: A critical review. J Conserv Dent 2013;16:284-93.  Back to cited text no. 2
[PUBMED]  [Full text]  
3.
Dohan DM, Choukroun J, Diss A, Dohan SL, Dohan AJ, Mouhyi J, et al. Platelet-rich fibrin (PRF): A second-generation platelet concentrate. Part II: platelet-related biologic features. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101:e45-50.  Back to cited text no. 3
    
4.
Dohan Ehrenfest DM, Pinto NR, Pereda A, Jiménez P, Corso MD, Kang BS, et al. The impact of the centrifuge characteristics and centrifugation protocols on the cells, growth factors, and fibrin architecture of a leukocyte- and platelet-rich fibrin (L-PRF) clot and membrane. Platelets 2018;29:171-84.  Back to cited text no. 4
    
5.
Tunalı M, Özdemir H, Küçükodacı Z, Akman S, Fıratlı E. In vivo evaluation of titanium-prepared platelet-rich fibrin (T-PRF): A new platelet concentrate. Br J Oral Maxillofac Surg 2013;51:438-43.  Back to cited text no. 5
    
6.
Ghanaati S, Booms P, Orlowska A, Kubesch A, Lorenz J, Rutkowski J, et al. Advanced platelet-rich fibrin: A new concept for cell-based tissue engineering by means of inflammatory cells. J Oral Implantol 2014;40:679-89.  Back to cited text no. 6
    
7.
Mourão CF, Valiense H, Melo ER, Mourão NB, Maia MD. Obtention of injectable platelets rich-fibrin (I-PRF) and its polymerization with bone graft: Technical note. Rev Col Bras Cir 2015;42:421-3.  Back to cited text no. 7
    
8.
Ghasemi A, Zahediasl S. Normality tests for statistical analysis: A guide for non-statisticians. Int J Endocrinol Metab 2012;10:486-9.  Back to cited text no. 8
    
9.
Banerjee B. Correlation and regression. In: Banerjee B, editor. Mahajain's methods in biostatistics for medical students and research workers. 9th ed. New Delhi: Jaypee Publishers; 2018. p. 249-69.  Back to cited text no. 9
    
10.
Miron RJ, Fujioka-Kobayashi M, Hernandez M, Kandalam U, Zhang Y, Ghanaati S, et al. Injectable platelet rich fibrin (I-PRF): Opportunities in regenerative dentistry? Clin Oral Investig 2017;21:2619-27.  Back to cited text no. 10
    
11.
Wend S, Kubesch A, Orlowska A, Al-Maawi S, Zender N, Dias A, et al. Reduction of the relative centrifugal force influences cell number and growth factor release within injectable PRF-based matrices. J Mater Sci Mater Med 2017;28:188.  Back to cited text no. 11
    
12.
Karde PA, Sethi KS, Mahale SA, Khedkar SU, Patil AG, Joshi CP. Comparative evaluation of platelet count and antimicrobial efficacy of injectable platelet-rich fibrin with other platelet concentrates: An in vitro study. J Indian Soc Periodontol 2017;21:97-101.  Back to cited text no. 12
[PUBMED]  [Full text]  
13.
Choukroun J, Ghanaati S. Reduction of relative centrifugation force within injectable platelet-rich-fibrin (PRF) concentrates advances patients' own inflammatory cells, platelets and growth factors: The first introduction to the low speed centrifugation concept. Eur J Trauma Emerg Surg 2018;44:87-95.  Back to cited text no. 13
    
14.
Aggarwal A, Singhal N. Evaluation of content and distribution of platelets in platelet rich fibrin at various centrifugation time periods: A light microscopic study. Int J Dent Med Res 2015;1:61-4.  Back to cited text no. 14
    
15.
Webb DI, Parker L, Webb K. Platelet count assessment from peripheral blood smear (PBS). Alaska Med 2004;46:92-5.  Back to cited text no. 15
    
16.
Varela HA, Souza JC, Nascimento RM, AraujVo RF, asconcelos RC, Cavalcante RS et al. Histological Preparation Technique of Blood Derivative Injectable Platelet-Rich Fibrin (I-Prf) for Microscopic Analyzes. J Cytol Histol 2018;9:2-5.  Back to cited text no. 16
    
17.
Melek LN, El Said MM. Evaluation of “autogenous bioengineered injectable PRF – tooth graft” combination (ABIT) in reconstruction of maxillary alveolar ridge defects: CBCT volumetric analysis. Saudi J Dent Res 2017;8:86-96.  Back to cited text no. 17
    


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  [Table 1], [Table 2]



 

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