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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 12  |  Issue : 1  |  Page : 22-26

Evaluation of L-platelet-rich fibrin in non- and post-COVID-19 patients and its role in periodontal regeneration – A microscopic analysis


1 Department of Periodontics and Implantology, College of Dental Sciences and Research Center Bopal, Ahmedabad, Gujarat, India
2 Department of Medical and Health Services, Deesa Civil hospital, Deesa, Gujarat, India

Date of Submission30-Jul-2021
Date of Decision30-Jul-2021
Date of Acceptance31-Aug-2021
Date of Web Publication31-Dec-2021

Correspondence Address:
Anita Panchal
Department of Periodontology and Implantology, College of Dental Sciences and Research Centre, Bopal, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/aihb.aihb_99_21

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  Abstract 


Introduction: Platelet-rich fibrin (PRF) is a term for autologous platelet concentrates generated from the patient's own blood (PRF). PRF and its derivatives (L-PRF, A-PRF, i-PRF) have been used for delicate tissue restoration in a variety of dental procedures. The quantity of leucocytes and other growth factors in PRF of healthy and post-COVID-19 people differs, according to the literature, and these influence wound tissue healing. Materials and Methods: Thirty healthy volunteers and 30 post-COVID-19 volunteers (age range 24–60 years). For PRF preparation, a REMI PR-23 table centrifuge and a blood collection kit consisting of a 19G needle and 10 ml blood collection tubes were used. The analysis was performed by dividing the subject groups into three test groups (Group 1 – post-COVID-19, 0–30 days; Group 2 – post-COVID-19, 31–90 days; Group 3 – normal patients). Group 1, 2 and 3 consisted of 23, 7 and 30 patients, respectively. Results: The result was statistically significant between the normal and posted COVID-19 patient groups (P = 0.00). Not much statistical significance was found between post-COVID-19 patients from 0–30 days to 31–90 days (P = 0.370). Considering the limitations of the study, our findings imply that typical patients' PRF clots or membranes comprise the majority of platelets and half of the leucocytes present in the first blood collection. Conclusion: Within the fibrin network, platelet growth factors are stuck, but the PRF clot or membrane of the post-COVID-19 patients contains a reduced/negligible number of leucocytes. Thus, the growth factors which is released are also less. Therefore, usage of PRF in post-COVID-19 patients for periodontal regenerative therapies should be avoided, at least for the first 60 days, to replenish the reduced leucocyte count and growth factors in the blood.

Keywords: COVID-19, leukocyte count, platelet-rich fibrin


How to cite this article:
Panchal A, Khan FA, Khan AH, Lakshmi P, Pandya MK, Pandya RK. Evaluation of L-platelet-rich fibrin in non- and post-COVID-19 patients and its role in periodontal regeneration – A microscopic analysis. Adv Hum Biol 2022;12:22-6

How to cite this URL:
Panchal A, Khan FA, Khan AH, Lakshmi P, Pandya MK, Pandya RK. Evaluation of L-platelet-rich fibrin in non- and post-COVID-19 patients and its role in periodontal regeneration – A microscopic analysis. Adv Hum Biol [serial online] 2022 [cited 2022 Jan 23];12:22-6. Available from: https://www.aihbonline.com/text.asp?2022/12/1/22/334571




  Introduction Top


Platelets, in addition to being involved in the inflammatory and immunological responses, may serve a novel and significant role in tissue repair and vascular remodelling, according to a new study.[1] Platelet creates naturally active proteins and other components that can influence a series of events, such as cell intake and growth etc., These substances may be secreted or exposed on the surface of stimulated platelets. The ability of platelets to discharge contents within a clot transforms the clot into a natural autologous source of growth factors and cytokines, which can be exploited to correctively speed up and healing processes will be accelerated.

Human platelet concentrate contains tiny fibrin fibres that can be detected by the high initial concentration of platelets (3–5 × 1011 platelets/l), the local procoagulant action might even be improved with the introduction of pro-thrombotic stimuli, and it encourages a practically harmful thrombin generation, resulting in an increase in fibrinogenesis on platelet surfaces, prompting fibrin development and polymerisation.[1]

Platelet-rich plasma and platelet-rich fibrin (PRP/PRF) was the pioneering step towards fulfilling clinical needs [Figure 1].[2] After single-step centrifugation without anticoagulants, a platelet-and leucocyte-rich framework was created from the patient's fringe blood. Platelets, leucocytes and fibrin have recently been shown to be essential for wound healing.[3],[4]
Figure 1: Different types of human platelet concentrates: Platelet-rich plasma; platelet-rich fibrin; pure platelet-rich plasma; leucocyte and platelet-rich plasma; pure platelet-rich fibrin); leucocyte and platelet-rich fibrin; injectable platelet-rich fibrin; advanced platelet-rich fibrin.[2]

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Furthermore, leucocyte's ability to influence angiogenesis and lymphomagenesis; this fibrin network, which contains leucocytes and platelets, is the foundation of cytokines and growth factors, which are primary membranes during the healing process.[5] The usage of plastic tubes benefits non-clotting PRF, and this resulted in the development of a fluid PRF-based network (fluid PRF) that was formed without the need for anticoagulants.

Furthermore, L-PRF (also known as leucocyte-PRF) contains white blood cells (WBC's), which are essential during the healing phase of an injury.[6] Furthermore, because WBC's, such as PMN and macrophages, are the first cells to arrive at wound sites, they play a role in phagocytosis of fragments, bacteria and necrotic tissue, preventing infection. Macrophages are essential cells produced from myeloid stem cells that are involved in the release of growth factors such as transforming growth factor-beta, platelet-derived growth factor and growth factor vascular endothelium during wound healing (VEGF) [Figure 2].[6]
Figure 2: Platelets play an important role in wound healing.[6]

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These cells, along with neutrophils and platelets, play an important role in wound healing, and their growth factors/released cytokines can aid tissue restoration, angiogenesis (the formation of new blood vessels) and infection prevention. Infections are prevented through phagocytosis and apoptosis.[3]

Therefore, considering the limitations, this study is to evaluate the L-PRF in non- and post-COVID-19 patients and its role in periodontal regeneration.


  Materials and Methods Top


Blood samples were collected at the College of Dental Sciences and Research Centre from 30 healthy patients and 30 post-COVID-19 patients (age range 24–60 years).

The following formula was used to determine the sample size.

N = (Zα/2 + Zβ) 2*2*σ2/d2.

(Where Z α/2 is the critical value of the normal distribution at α/2). The samples were collected from resident doctors of college and patients that came for a routine dental check-up. The ethical clearance for this study with reference no. CD5/Admin. 096/A/20 on 25/08/2020 was obtained from the College of Dental Sciences and Research Centre. The equipment required for PRF preparation includes a REMI PR-23 table centrifuge and a blood collection kit consisting of a 19-gauge needle and 10 ml blood collection tubes.

A sample of blood is collected from the patient without anticoagulant in 10 ml tubes which are immediately centrifuged to prepare PRF according to Choukroun's method.[7] During the centrifugation process, when the blood gets in contact with the test tube wall, the platelet gets activated, leading to the initiation of the coagulation cascade.

The 10 ml of blood was collected rapidly with needle 19-G to vacutainer tubes and quickly (within 1 min) centrifuged. The outcome product is made of three membranes: Platelet-poor plasma at the top, PRF (central clot), red blood cells (RBCs) at the base [Figure 1].

Resulting PRF clots are assembled, and RBCs are expelled with the aid of scissors, without macroscopical damage at PRF structure expense. There is no macroscopic damage to the membrane as the cut is made about 2 mm beyond the white clot.

Histological procedure

Macroscopic procedure

After centrifugation, the PRF clot was removed from the tube using sterile tweezers and a smooth spatula to gently release the RBCs clot inside the tube [Figure 3]c. The PRF fibrin clot obtained was placed on a sterile microscope slide [Figure 3]a and [Figure 3]b. The supernatant and RBCs clots remaining in the tube were also weighted to get the PRF fibrin clot/whole blood ratio per tube.
Figure 3: (a and b) The PRF placed in between the sterile microscope slide, (c) AUTOLOGOUS PRF

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Each sterile microscopic slide allowed the compression of the clot with another microscopic slide using constant pressure for 2 min. This standardised method allowed to obtain from each clot 1 mm-thick PRF membranes.

From each volunteer (post-COVID and normal patient), two membranes were obtained per centrifuge. The membrane was prepared for the light-microscopy analysis. The membranes were kept between the microscope slides during fixation to avoid distortions.

Light microscopy procedure

The routine procedure was taken out i.e., Fixation, Tissue processing, Embedding, Tissue sectioning, Dewaxing the slides, Tissue staining, and then a light microscope with a magnification of 400x was used to examine histological slides in which we can observe in [Figure 4]b, leucocytes were lesser/absent, which contradict the non-covid patient's PRF in [Figure 4]a consisting a greater number of them.
Figure 4: (a) Non-COVID. (b) Post-COVID

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Statistical analysis

The mean and standard deviation were used to represent all of the data. To examine the mean difference between the leucocyte present in healthy and post-COVID person's PRF membranes, a one-way ANOVA with Scheffe post-hoc test with multiple comparisons was used. The IBM SPSS software 21 Banglore, Karnatka and India was used to analyse the data.


  Result Top


Microscopic analysis

A total of 60 samples were collected (30 from normal person and 30 from post-COVID-19 patients). On comparing this PRF membrane, it was found that the mean leucocyte count for Group 1, which includes the patients with post-COVID-19 from 0 to 30 days, is 0.26 for, Group 2 which include patients with post-COVID-19 from 31 to 90 days, is 1.43 and for Group 3 which was not affected by COVID-19 is 10.27 with the standard deviation of 0.449, 1.134, 2.586, respectively. [Table 1] shows the mean leucocyte count of the three groups, and it shows a statistically significant difference between these three groups. Scheffe correlation was done to compare the leucocyte difference between the groups, and the result was statistically significant, as shown in [Table 2].
Table 1: The number of leukocytes in the control and test groups after collecting the platelet-rich fibrin membrane

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Table 2: Scheffe association between the three group's leucocyte counts

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  Discussion Top


COVID-19 is a serious disease in which patients develop respiratory diseases and multiple organ failure rapidly, resulting in death within a short time. Cytokine storm is considered to be one of the major causes of ARDS and multiple organ failure. It plays a crucial function in the progression of the disease. A cytokine storm has been found in COVID-19-positive critical patients in clinical investigations.

COVID-19 patients with a wide range of clinical symptoms have been found to have lymphocytopenia, albeit the alterations in distinct lymphoid subsets have yet to be determined. In a previous study, mainly in noncritical patients infected with SARS-CoV-2, 35% of the patients had only mild lymphocytopenia. Lymphocytopenia occurred in more than 80% of the severe COVID-19 patients.[8]

The purpose of the study is to determine the changes in the leucocyte count in normal and post-COVID-19 patient's PRF.

A total number of 60 patients were included in the study. The age group was ranging from 24 to 60 years. In COVID-19 patients, many studies have identified an independent link between the male sex and poorer outcomes.[9] However, in this study, the clinical severity of the illness was not associated with the sex of the patient.

The samples were divided into two groups, the test group being a measurement of leucocytes in PRF of 30 patients who had contracted and recovered from COVID-19. The control group consisted of 30 patients who had not been previously exposed to or suffering from COVID-19.

Blood was collected from the patients without anticoagulant in 10 ml tubes which were immediately centrifuged according to Choukroun's method.[7] The blood of 10 ml was collected rapidly with needle 19-G to vacutainer tubes and quickly (within 1 min) centrifuged. Leucocyte's number in the control group and test group after collecting the PRF membrane has been listed in [Table 1]. The mean differential leucocyte count was 5.40 ± 5.263 cells/mm3.

The analysis was done by dividing the subject groups into three test groups (1 – post-COVID-19, 0–30 days; 2 – post-COVID-19, 31–90 days; 3 – normal patients). Group 1, 2 and 3 consisted of 23, 7 and 30 patients, respectively. Scheffe correlation with multiple comparisons was done to compare the leucocyte difference between Groups 1 and 2 and 2 and 3. P was set to be at 0.05. The Scheffe correlation comparing the leucocyte count in the three groups has been described in [Table 2].

Systemic illness like diabetes and hypertension has a direct relation on leucocyte count and sedimentation rate. To follow the purpose of the study and to only check for the correlation of leucocyte count before and after exposure to COVID-19, patients having co-morbidities such as diabetes or hypertension were excluded from the study.

Not much statistical significance was observed between Groups 1 and 2 (1.168 ± 0.821, P = 0.370). However, a recent study showed significant differences in leucocyte count and cytokine storm between mild and severe manifestations of COVID-19. This can have a direct correlation between the recuperation rate of the exhausting number of leucocytes and the severity of COVID-19.[10]

Values were statistically significant between - Group 2 and 3 (10.006 ± 0.527, P = 0.00). This is in line with a study that found that COVID-19 pneumonia has lower leucocyte, PMN and platelet counts than non-COVID-19 pneumonia.[11]

A study also reported that patients that there is decreased lymphocyte/WBC ratio. Unlike non-survivors, survivors showed a trough in lymphocyte count on day seven after symptom start, followed by a restoration.[12]

As a result, monitoring lymphocyte count dynamics over time may be a good predictor of patient outcome. Patients with <20% lymphocytes at days 10–12 after the beginning of symptoms and <5% lymphocytes at days 17–19 have the worst prognosis, according to Tan et al.[13]

The cause of lymphopenia in patients is yet unclear. Given that lymphocytes express low levels of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for both SARS-CoV-2 and SARS-CoV, and the viral genome is rarely detectable in SARS-CoV-2 infected patients' peripheral blood, it is reasonable to assume that the decrease in peripheral lymphocytes is not directly attributed to SARS-CoV-2 infection.

Another theory is that the disease of peripheral lymphocytes is caused by activation-induced apoptosis or aggressive migration from the peripheral circulation to the lungs, where the virus replicates vigorously. Further research is needed to determine the processes that cause lymphopenia. CD4+ T-cells were observed to be significantly reduced in the peripheral blood of severe SARS-CoV-2 infected individuals.[14]

Because of the SARS-CoV-2 virus, the immunity level of the person becomes low. Thus, the healing capacity of the PRF would be low due to the low growth factors released.[15]


  Conclusion Top


Platelets are mainly active and function as cement to support the fibrin matrix, which is highly polymerised. The bulk of lymphocytes (leukocytes) are stuck within this fibrin network, yet they are still active and ready to migrate in culture. Within the fibrin network, platelet growth factors are stuck, but the PRF clot or membrane of the post-COVID-19 patients contains a reduced/negligible number of leucocytes. Thus, the growth factors which is released are also less. Therefore, the PRF membrane of post-COVID-19 patient's capacity to regenerate during wound healing would be less. Usage of PRF in post-COVID-19 patients for periodontal regenerative therapies should be avoided, at least for the first 60 days, to replenish the reduced leucocyte count and growth factors in the blood.

Acknowledgements

The authors express their thanks to Dr Madhukant Shah, Dr. Hitendra Shah, Dr. Suchi Shah, Dr. Binita Gandhi, Deepakbhai, Soyab Mandori, Dr. Falguni Gor, Dr. Harshvardhan Chaudhary.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Pretorius E, Briedenhann S, Marx J, Smit E, Van Der Merwe C, Pieters M, et al. Ultrastructural comparison of the morphology of three different platelet and fibrin fiber preparations. Anat Rec (Hoboken) 2007;290:188-98.  Back to cited text no. 1
    
2.
Choukroun J, Adda F, Schoeffler C, Vervelle A. An opportunity in paro-implantology: The PRF. Implantodontics 2001;42:55-62.  Back to cited text no. 2
    
3.
Crisci A, De Crescenzo U, Crisci M. Platelet-Rich Concentrates (L-PRF, PRP) in tissue regeneration: Control of apoptosis and interactions with regenerative cells. J Clin Mol Med 2018;1:5-12.  Back to cited text no. 3
    
4.
Litvinov RI, Weisel JW. What is the bioscientific and clinical relevance of fibrin? Semin Thromb Hemost 2016;42:333-43.  Back to cited text no. 4
    
5.
Soloviev DA, Hazen SL, Szpak D, Bledzka KM, Ballantyne CM, Plow EF, et al. Dual role of the leukocyte integrin αMβ2 in angiogenesis. J Immunol 2014;193:4712-21.  Back to cited text no. 5
    
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Crisci A, Lombardi D, Serra E, Lombardi G, Cardillo F, Crisci M. Standardized protocol proposed for clinical use of L-PRF and the use of L-PRF Wound Box®. J Unexplored Med Data 2017;2:77-87.  Back to cited text no. 6
    
7.
Dohan DM, Choukroun J. PRP, cPRP, PRF, PRG, PRGF, FC. How to find your way in the jungle of platelet concentrates? Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;103:305-6.  Back to cited text no. 7
    
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Ye Q, Wang B, Mao J. The pathogenesis and treatment of the 'Cytokine Storm' in COVID-19. J Infect 2020;80:607-13.  Back to cited text no. 8
    
9.
Takahashi T, Ellingson MK, Wong P, Israelow B, Lucas C, Klein J, et al. Sex differences in immune responses that underlie COVID-19 disease outcomes. Nature 2020;588:315-20.  Back to cited text no. 9
    
10.
Green MS, Peled I, Najenson T. Gender differences in platelet count and its association with cigarette smoking in a large cohort in Israel. J Clin Epidemiol 1992;45:77-84.  Back to cited text no. 10
    
11.
Song JW, Zhang C, Fan X, Meng FP, Xu Z, Xia P, et al. Immunological and inflammatory profiles in mild and severe cases of COVID-19. Nat Commun 2020;11:3410.  Back to cited text no. 11
    
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Usul E, Şan İ, Bekgöz B, Şahin A. Role of hematological parameters in COVID-19 patients in the emergency room. Biomark Med 2020;14:1207-15.  Back to cited text no. 12
    
13.
Tan L, Wang Q, Zhang D, Ding J, Huang Q, Tang Y-Q, et al. Lymphopenia predicts disease severity of COVID-19: A descriptive and predictive study. Signal Transduct Target Ther 2020;5:33.  Back to cited text no. 13
    
14.
Bhardwaj A, Sapra L, Saini C, Azam Z, Mishra PK, Verma B, et al. COVID-19: Immunology, immunopathogenesis and potential therapies. Int Rev Immunol 2021;27:1-36.  Back to cited text no. 14
    
15.
Long QX, Tang XJ, Shi QL, Li Q, Deng HJ, Yuan J, et al. Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nat Med 2020;26:1200-4.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2]



 

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