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Authors: Dr. Anshuman Dwivedi
 

The topical use of platelet concentrates is recent and its efficiency remains controversial. Several techniques for platelet concentrates are available; however, their applications have been confusing because each method leads to a different product with different biology and potential uses.1 The biological activity of platelet concentrates differs according to preparation technique, which affects platelet and leukocyte content and GF availability.2 The wide variation of reported protocols for preparation of PRP leads to variable compositions, which induce different biological responses and prevent results comparison.4 The classification of the different platelet concentrates, depending on their leucocyte and fibrin content is as follows :1

  1. pure platelet-rich plasma (P-PRP), such as cell separator PRP, Vivostat PRF or Anitua's PRGF; (fig 1)
  2. leucocyte- and platelet-rich plasma (L-PRP), such as Curasan, Regen, Plateltex, SmartPReP, PCCS, Magellan or GPS PRP; (fig 2,fig 3)
  3. pure plaletet-rich fibrin (P-PRF), such as Fibrinet;
  4. leucocyte- and platelet-rich fibrin (L-PRF), such as Choukroun's PRF. (fig 4)
fig 1: PRGF clot after mixing autogenous bone obtained during osteotomy and activation
 
fig 2 Processing of whole blood (retrieved from http://www.coequine.com/prp.html on 25th oct 2014 at 22:50 )
 
fig 3.Separation of platelet rich plasma from platelet poor plasma
(retrieved from http://t0.gstatic.com/images?q=tbn:ANd9GcQEdwPPKPr
jS9gu-SpdFm0hEIoM6_gl1_PmNlQUBGdkNWFLMQ6-ZPqBwuA on 25th
Oct 2014 at 22:43 )
fig 3.Separation of platelet rich plasma from platelet poor plasma (retrieved from http://t0.gstatic.com/images?q=tbn:ANd9GcQEdwPPKPr jS9gu-SpdFm0hEIoM6_gl1_PmNlQUBGdkNWFLMQ6-ZPqBwuA on 25th Oct 2014 at 22:43 )

fig 4: PRF prepared after centrifugation

Centrifugation is one of the most widely used processes in liquid-liquid or solid-liquid separation. It is based on the application of a centrifugal force that is much higher than gravity. The difference in size and density of the particles in the various phases is the driving force responsible for the separation. During the process of centrifugation, the movement of the particle is a result of the acting centrifugal force in the radial direction, the gravitational force in the downward direction, and the drag force in the opposing direction of particle motion. All the mentioned forces are quickly balanced. The magnitude of the centrifugal force acting depends on the apparent mass of the particle (corrected to the buoyancy), the angular velocity, and its distance from the axis of the centrifugal head or rotor. The greater the distance from the rotor, the greater the centrifugal force acting on the particle.4 the factors affecting centrifugation are-

  1. Time: According to the physics of the centrifugation process, time and acceleration are the fundamental parameters that define the composition of the Platelet concentrate sample. Longer time periods slightly increased platelet recovery and decreased the concentrations of WBC in the upper layer.4
  2. Processed Volume. The processing of a larger volume of whole blood (8.5 mL) in a single tube decreased the recovery efficiency of platelets and the packing of the erythrocytes compared to 3.5mL of whole blood processed at the same conditions. Therefore, in this situation, the centrifugal acceleration and time should be adjusted to achieve the same packing of erythrocytes and the recovery of platelets and plasma as a consequence.4
  3. Speed. The initial plasma concentrations of sP-selectin for two of the donors ranged from18 to 40 ng/mL.These values are considered to be in the normal range of concentration values for human plasma . Figure 5 shows that the concentration of sP-selectin from both donors effectively increased when the centrifugal acceleration was 800 ×g and 1200×g, indicating activation of the platelets during centrifugation (herein considered as loss of platelet integrity).4

Platelet Concentration Gradient. Platelet concentration gradients are formed after spins. Various factors contribute to this gradient such as the sizes of platelets, from the range of peripheral platelets as measured in femtoliters (1015 L), until the biological difference among individuals along with haematocrit variability The presence of remaining RBC can generate a pellet at the bottom of the tube, which adsorb platelets and WBC on its surface, as evidenced experimentally. The manual mixing for a short period of time was insufficient to completely resuspend the platelets and large variability in platelet counting was observed. The samples mechanically mixed by tube inversion for 30 or 60 min had an improved recovery efficiency of platelets (75–80%) regardless of the centrifugal acceleration that was applied. Thus, approximately 20% of platelets remained adsorbed in the RBC pellet. According to Turkey’s test ( =0.05), there is no statistical differences for 30 or 60min mixture. The residual of platelets was minimal when centrifugal accelerations of 400 ×g or higher were applied.

fig 5 Effects of centrifugal acceleration on concentration of sP-selection after the second spin.

(Amanda G et.al; Relevant Aspects of Centrifugation Step in the Preparation of Platelet-Rich Plasma ; Hindawi Publishing Corporation ISRN Hematology Volume 2014)

In the case of whole blood, the centrifugal force and time drive the packing of erythrocytes at the bottom layer, the volume of plasma at the upper layer, and the recovery efficiency of platelets. Anitua et al.6 used only one centrifugation spin step and collected the volume immediately above the erythrocyte layer. This protocol obtained a platelet concentration factor of 2.67 above the baseline value. When all of the volume of the upper layer is collected, regardless of whether the buffy coat layer is included, additional spins can be performed to achieve higher platelet concentration factors (>3×) 7,8. Kahn et al. (1976) determined that a centrifugal acceleration of 3730 ×g for a period of 4 min was the optimal condition for obtaining the highest platelet concentration from 478mL of Whole blood 9. The highest platelet recovery efficiency obtained by Slichter and Harker (1976) was 80%, using a sample of 250–450mL of WB centrifuged at 1000 ×g for a period of 9min10 .It was observed that a subsequent centrifugation step of 3000 ×g for a period of 20min decreased the platelet viability. Bausset et al.11 found that a centrifugation of 130 or 250 ×g for a period of 15min was optimal when performing a procedure that involved two spins. A platelet concentration factor of 3.47 was obtained from the 8.5mL whole blood processed, and 2.0mL of plasma was processed in the second spin step. Although different methods were used, the authors evaluated the platelet integrity, and the data are consistent with those of our study. Mazzocca et al. 12 analyzed 3 protocols for preparing PRP samples with different compositions: a low platelet (382×103 /mm3) and low WBC (0.6 × 103 /mm3) process with one spin step at 1500 rpm for 5min (10mL whole blood); a high platelet (940 × 103/mm3) and high WBC (17 × 103/mm3) process with one spin step at 3200 rpm for 15min (27mL whole blood); and a double-spin process (1500 rpm for 5min and 6300 rpm for 20 min) that produced a higher platelet concentration (472 ×103/mm3) and lower WBC (1.5 × 103/mm3). Many other studies13, 14 specify centrifugal accelerations in rotations per minute (RPM) instead of in ×g, complicating the task of comparing and reproducing their results.

CONCLUSIONS

Centrifugal acceleration, time, distance between the particles and the rotor, the volume of processed whole blood, prevention of platelet aggregation, and minimization of platelet gradient before measurements are the main relevant aspects to be controlled in the centrifugation step for the preparation and characterization of platelet concentrate with maximum benefits. The observance of these aspects ensures the overall quality of platelet concentrate by allowing the variability of results to become restricted only to the autologous nature of the product. This is the starting point for comparison of biological results as well as for the standardization of the platelet concentrate for specific applications in vivo.

REFERENCES
  1. David M. Dohan Ehrenfest, Lars Rasmusson, and Tomas Albrektsson;Classification of platelet concentrates from pure platelet-rich plasma (P-PRP) to leukocyte- and platelet-rich fibrin (L-PRF); Cytotherapy. 2013 Jul; 15(7):830-9. doi: 10.1016/j. jcyt.2013.01.220
  2. Perut F et.al, ;Preparation method and growth factor content of platelet concentrate influence the osteogenic differentiation of bone marrow stromal cells; International Society for Cellular Therapy.2013
  3. Jun Araki, M.D. et.al ; Optimized Preparation Method of Platelet-Concentrated Plasma and Non coagulating Platelet-Derived Factor Concentrates: Maximization of Platelet Concentration and Removal of fibrinogen ; TISSUE ENGINEERING: Part C Volume 18, Number 3, 2012DOI: 10.1089/ten.tec.2011.0308
  4. Amanda G et.al; Relevant Aspects of Centrifugation Step in the Preparation of Platelet-Rich Plasma ; Hindawi Publishing Corporation ISRN Hematology Volume 2014
  5. Beth Callan, VMD, Frances S. Shofer, PhD, and James L. Catalfamo, PhD;Effects of anticoagulant on pH, ionized calcium concentration, and agonist-induced platelet aggregation in canine platelet-rich plasma ;Mary Am J Vet Res. Apr 2009; 70(4): 472–477.
  6. E. Anitua, J. J. Aguirre, J. Algorta et al., “Effectiveness of autologous preparation rich in growth factors for the treatment of chronic cutaneous ulcers,” Journal of Biomedical Materials Research B: Applied Biomaterials, vol. 84, no. 2, pp. 415–421,2008.
  7. C. H. Jo, Y. H. Roh, J. E. Kim, S. Shin, K. S. Yoon, and J. H. Noh,“Optimizing platelet-rich plasma gel formation by varying time and gravitational forces during centrifugation,” The Journal of Oral Implantology, vol. 39, no. 5, pp. 525–532, 2013.
  8. R. Landesberg, M. Roy, and R. S. Glickman, “Quantification of growth factor levels using a simplified method of plateletrich plasma gel preparation,” Journal of Oral andMaxillofacial Surgery, vol. 58, no. 3, pp. 297–300, 2000.
  9. R. A. Kahn, I. Cossette, and L. I. Friedman, “Optimum centrifugation conditions for the preparation of platelet and plasma products,” Transfusion, vol. 16, no. 2, pp. 162–165, 1976.
  10. S. J. Slichter and L. A. Harker, “Preparation and storage of platelet concentrates. I. Factors influencing the harvest of viable platelets fromwhole blood,” British Journal of Haematology, vol.34, no. 3, pp. 395–402, 1976.
  11. O. Bausset, L. Giraudo, J. Veran et al., “Formulation and storage of platelet-rich plasma homemade product,” Biores Open Access, vol. 1, no. 3, pp. 115–123, 2012.
  12. A. D. Mazzocca, M. B. R. McCarthy, D. M. Chowaniec et al.,“Platelet-rich plasma differs according to preparation method and human variability,”The Journal of Bone & Joint Surgery A,vol. 94, no. 4, pp. 308–316, 2012.
  13. 13. J. E. Fern´andez-Barbero, P. Galindo-Moreno, G. ´ Avila-Ortiz, O.Caba, E. S´anchez-Fern´andez, and H.-L. Wang, “Flow cytometric and morphological characterization of platelet-rich plasma gel,” Clinical Oral Implants Research, vol. 17, no. 6, pp. 687–693,2006.
  14. 14. C. Y. Su, Y. P. Kuo, H.-L. Nieh, Y. H. Tseng, and T. Burnouf,“Quantitative assessment of the kinetics of growth factors release from platelet gel,” Transfusion, vol. 48, no. 11, pp. 2414–2420, 2008.

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