Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination

Platelet-rich fibrin (PRF) clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP) fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

[1]  T. Kawase,et al.  Striking Differences in Platelet Distribution between Advanced-Platelet-Rich Fibrin and Concentrated Growth Factors: Effects of Silica-Containing Plastic Tubes , 2019, Journal of functional biomaterials.

[2]  M. Neagu,et al.  Alveolar blood clots and platelet-rich fibrin induce in vitro fibroblast proliferation and migration , 2018, Experimental and therapeutic medicine.

[3]  Kohya Uematsu,et al.  An on‐site preparable, novel bone‐grafting complex consisting of human platelet‐rich fibrin and porous particles made of a recombinant collagen‐like protein , 2018, Journal of biomedical materials research. Part B, Applied biomaterials.

[4]  T. Kawase,et al.  Spectrophotometric determination of platelet counts in platelet-rich plasma , 2018, International Journal of Implant Dentistry.

[5]  Tomoyuki Kawase,et al.  Comprehensive Quality Control of the Regenerative Therapy Using Platelet Concentrates: The Current Situation and Prospects in Japan , 2018, BioMed research international.

[6]  T. Kawase,et al.  An updated proposal for terminology and classification of platelet-rich fibrin , 2017, Regenerative therapy.

[7]  T. Kawase,et al.  Quality Assessment of Platelet-Rich Fibrin-Like Matrix Prepared from Whole Blood Samples after Extended Storage , 2017, Biomedicines.

[8]  Kohya Uematsu,et al.  Mechanical and degradation properties of advanced platelet-rich fibrin (A-PRF), concentrated growth factors (CGF), and platelet-poor plasma-derived fibrin (PPTF) , 2017, International journal of implant dentistry.

[9]  R. Miron,et al.  Basics of Platelet-Rich Fibrin Therapy. , 2017, Dentistry today.

[10]  S. Ghanaati,et al.  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 , 2017, European Journal of Trauma and Emergency Surgery.

[11]  Taiji Suzuki,et al.  Platelet-rich fibrin prepared from stored whole-blood samples , 2017, International Journal of Implant Dentistry.

[12]  R. Jacobs,et al.  Platelet-rich plasma for regeneration of neural feedback pathways around dental implants: a concise review and outlook on future possibilities , 2017, International Journal of Oral Science.

[13]  Taiji Suzuki,et al.  An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations , 2017, Dentistry journal.

[14]  R. Miron,et al.  Platelet-Rich Fibrin and Soft Tissue Wound Healing: A Systematic Review. , 2016, Tissue engineering. Part B, Reviews.

[15]  M. Quirynen,et al.  The impact of autologous platelet concentrates on endodontic healing: a systematic review , 2016, Platelets.

[16]  Kohya Uematsu,et al.  Growth factor and pro-inflammatory cytokine contents in platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), advanced platelet-rich fibrin (A-PRF), and concentrated growth factors (CGF) , 2016, International journal of implant dentistry.

[17]  S. Varghese,et al.  Additive effect of autologous platelet concentrates in treatment of intrabony defects: a systematic review and meta-analysis. , 2016, Journal of investigative and clinical dentistry.

[18]  H. Atmaca,et al.  Effect of centrifugation time on growth factor and MMP release of an experimental platelet-rich fibrin-type product , 2016, Platelets.

[19]  T. Kawase,et al.  In vitro immunological and biological evaluations of the angiogenic potential of platelet-rich fibrin preparations: a standardized comparison with PRP preparations , 2015, International journal of implant dentistry.

[20]  Takashi Inoue,et al.  The Effect of Concentrated Growth Factor on Rat Bone Marrow Cells In Vitro and on Calvarial Bone Healing In Vivo. , 2015, The International journal of oral & maxillofacial implants.

[21]  E. Borie,et al.  Platelet-rich fibrin application in dentistry: a literature review. , 2015, International journal of clinical and experimental medicine.

[22]  T. Kawase.  Platelet-rich plasma and its derivatives as promising bioactive materials for regenerative medicine: basic principles and concepts underlying recent advances , 2015, Odontology.

[23]  T. Kawase,et al.  The heat-compression technique for the conversion of platelet-rich fibrin preparation to a barrier membrane with a reduced rate of biodegradation. , 2015, Journal of biomedical materials research. Part B, Applied biomaterials.

[24]  G. Orive,et al.  Leukocyte Inclusion within a Platelet Rich Plasma-Derived Fibrin Scaffold Stimulates a More Pro-Inflammatory Environment and Alters Fibrin Properties , 2015, PloS one.

[25]  K. Fujita,et al.  Growth Factor Measurement and Histological Analysis in Platelet Rich Fibrin: A Pilot Study , 2015, Journal of Maxillofacial and Oral Surgery.

[26]  A. Aggarwal,et al.  Evaluation of Content and Distribution of Platelets in Platelet Rich Fibrin at Various Centrifugation Time Periods: A Light Microscopic Study , 2015 .

[27]  Sanjeev Palta,et al.  Overview of the coagulation system , 2014, Indian journal of anaesthesia.

[28]  M. H. Santana,et al.  Fibrin network architectures in pure platelet-rich plasma as characterized by fiber radius and correlated with clotting time , 2014, Journal of Materials Science: Materials in Medicine.

[29]  E. Kruithof,et al.  Human tissue-type plasminogen activator , 2014, Thrombosis and Haemostasis.

[30]  T. Kawase,et al.  A proposed protocol for the standardized preparation of PRF membranes for clinical use. , 2012, Biologicals : journal of the International Association of Biological Standardization.

[31]  D. M. Dohan Ehrenfest,et al.  Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane. , 2010, Journal of periodontology.

[32]  C. Su,et al.  In vitro release of growth factors from platelet-rich fibrin (PRF): a proposal to optimize the clinical applications of PRF. , 2009, Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics.

[33]  I. Springer,et al.  Platelet-rich plasma and platelet-rich fibrin in human cell culture. , 2009, Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics.

[34]  E. Vogler,et al.  Contact activation of blood-plasma coagulation. , 2009, Biomaterials.

[35]  M. Ågren,et al.  Bioactivity and stability of endogenous fibrogenic factors in platelet‐rich fibrin , 2008, Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society.

[36]  A. Dohan,et al.  Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution. , 2006, Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics.

[37]  K. Kvell,et al.  Flow cytometric analysis of CD41‐labeled platelets isolated by the rapid, one‐step OptiPrep method from human blood , 2005, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[38]  E. Freymiller.  Platelet-rich plasma: evidence to support its use. , 2004, Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons.

[39]  W. Nieuwenhuizen.  Fibrin‐Mediated Plasminogen Activation , 2001, Annals of the New York Academy of Sciences.

[40]  R. Marx,et al.  Platelet-rich plasma (PRP): what is PRP and what is not PRP? , 2001, Implant dentistry.

[41]  Michael R. Groves,et al.  Application of the Electrical Sizing Principle of Coulter to a New Multiparameter System , 1980, IEEE Transactions on Biomedical Engineering.