COMPARISON OF DIFFERENT METHODS OF CENTRIFUGATION FOR OPTIMUM PREPARATION OF PLATELET RICH PLASMA (PRP)& FRAMING A CUSTOMIZED PRP PROTOCOL-AN INNOVATIVE APPROACH

Introduction: The rationale behind the therapeutic potential of the high of platelets of is by the ability of the centrifugation followed in the high study aims to the basic of PRP preparation involved in centrifugation protocols, spin methods, speed and time duration of centrifugation to obtain consistent therapeutic platelets yield. Materials and Methods :30 participants were subjected to intervention procedures using PRP. For therapeutic purpose 3 centrifugation protocol types are followed.


Aim
The aim of the present study is to compare the biological characteristics of PRP focusing on the platelet & leukocyte concentration and composition, derived out of different centrifugation methods from a physiatry view. Objectives:-

Operational procedure
Whole blood is initially collected in tubes that contain anticoagulant Acid Citrate Dextrose (preserves platelet viability) in the ratio of 6:1. [11] The first spin step is performed at constant acceleration to separate RBCs from the remaining WB volume. After the first spin step, the WB separates into three layers: an upper layer that contains platelets poor plasma, an intermediate layer that is known as the buffy coat and that is rich in WBCs, and a bottom layer that consists mostly of RBCs. Only the upper layer or the upper layer plus buffy coat is transferred to an empty sterile vacutainer.
66 Formula RCF=1.12*R(S/1000) 2 *gWhere "g" is the effect of earth"s gravitation field; RCF (Relative Centrifugal Force), R is the radius of the rotor (from center of rotor to sample) in millimeters and S is the speed of the centrifuge in revolutions per minute. [4] Radius of Rotor-125 mm 67 Ispin-100g=850 rpm (FOR PLATELET SEPARATION) [9] II spin-400g =1700 rpm (FOR PLATELET CONCENTRATION) [9] Note: -100 g and 400 g respectively for first and second spins are already proposed nominal values that have yielded better results in previous studies. [9] As per the centrifugal acceleration force generated is represented in terms of g force (effect of earth"s gravitation field) than RPM, [9] the new spin methods (PROTOCOL 2) calculated according to the formula and the protocol are used to harvest autologous PRP and the concentration factor and recovery efficiency of platelets yielded are compared with the old conventional centrifugationmethod (PROTOCOL1). Likewise, the WBC concentrations are also compared between the two spin methods. Since literature recommends [13] spin reversal (hard spin followed by soft spin for better WBC yield in PRP), that is also tried.

Results:-
Of the 30 participants,19 participants are female and 11 are male with mean age of 52.23 years.8 followed the old spin protocol,8 followed new spin protocol and 14 followed the spin reversal technique.10 underwent intra articular knee joint injections. 16     As determined by one-way ANOVA, there was a statistically significant difference between the PROTOCOL groups (1,2,3) for the variables PRP platelet count, PRP LEUCOCYTES, PLATELET CONCENTRATION FACTOR and PLATELET RECOVERY RATIO whereas, there was no statistically significant difference for WB platelet count and WB LEUCOCYTES variables. Table 5:-Post Hoc (Tukey HSD).

Whole Blood Vs PRP Platelets Graph 1:-Platelet concentration Range.
Inference:-A Tukey post hoc test revealed that there was statistically significantly lower for PROTOCOL 2 and PROTOCOL 3 compared to the PROTOCOL 2 forPRPplateletcount, PLATELETCONCENTRATIONFACTOR and PLATELETRECOVERYRATIO whereas for PRPLEUCOCYTES, there was a statistically significant difference between the PROTOCOL 2 and PROTOCOL 3 which again implicates the necessity of spin reversal in yielding better quantity of leucocytes.
A Tukey post hoc test also revealed that there was no statistically significant difference between the PROTOCOL 1 and PROTOCOL 3 for PRP platelet count, PLATELET CONCENTRATION FACTOR and PLATELET At least more than half of the outcome variables assessed have showed significant statistical difference by ANOVA and there is statistically significant difference between protocol 1*3 and protocol 1*2.but not between 2&3 by post hoc Tukey test. Hence the alternate hypothesis of customisation of new centrifugation protocol based on rationalisation of parameters shall be accepted and expected to set a standard to enhance the quality of PRP optimally. Figure 6:-PRP yield as per literature The temperature in which centrifugation is carried out Leucocyte rich PRP is meant for articular cartilages /structures with type2 collagen Leucocyte poor PRP gives better result in places of tendons/structures with type 1 collagen [20] What we learnt from the study Double spin is better than single spin of centrifugation (single spin would not produce a true PRP. Instead, a mixture of PRP and PPP with disappointingly low platelet counts. will be produced). [3] 1. I Spin-platelet separation 2. II spin-platelet concentration 3. Variations in the volume of blood processed (5 to 120 ml) 4. Centrifugation shall be customized according to the centrifuge used 5. RPM (rotations per minute)-the term is now being replaced by g force 6. Customized preparation of PRP`~-Commercial PRP kits 7. Processing Qualitative Standards (PQLSs)-Centrifugation speed, duration, anticoagulants, temperature and the type of centrifuge and technical precision play a major role in determining the fruitful clinical outcome [15] 8. Platelets may get distorted at higher speed affecting extrapolation of clinical and experimental results [9] Discussion:-According to the physics of the centrifugation process, time and acceleration are the fundamental parameters that define the composition of the PRP sample after the first spin step. [16] According to the physical behavior, the extrapolation of the operating parameters is not straight forward, since it involves an exponential relationship with the distance traveled by the particles in the centrifugation. The method for quality assurance of individual PRP preparations should be given weightage equally and assessed. Still there are few unknowns/least considered facts regarding the processes that may have an impact on treatment efficacy for musculoskeletal conditions being dealt by the Physical Medicine and Rehabilitation team [17] The high concentrations of cells are important, as the white blood cell count in PRP samples has frequently been ignored, being considered insignificant. These findings demonstrate that leukocytes strongly influence the quality of PRPs. [19] Therefore, modifying the PRP preparation method according to the pathology is essential to achieve better clinical results with PRP therapy. There is currently disagreement in the literature over whether the presence of WBC in PRP provides any benefit. Proponents of PRP containing high WBC concentrations (Leukocyte and  73 Platelet-Rich Plasma (L-PRP) according to Ehrenfest classification believe that the presence of WBC provides natural protection against infections and allergic responses [13] 1. The minimum PRP platelet count is from old spin method (PROTOCOL 1) 2. The minimum PRP WBC is from both old and new spin methods which in turn depicts that reversal of spins favors increased WBC yield. 3. The minimum PCF and PRR is from new customized spin. (PROTOCOL 2) 4. The maximum PRP platelet and WBC count is from spin reversal method and the same has been reflected in the mean values too. 5. The maximum PCF and PRR is from PROTOCOL 1but the mean values are maximum through PROTOCOL 2 Which recommends that customization yields better quantity platelets. 6. The lowest mean of PRP Platelets and WBC, PCF, PRR are from old spin method. 7. The maximum PCF-3.21 times above baseline and PRR -64.21% are through new customized spin proving its efficiency 8. The results are variable in PRP method and buffy coat method and the platelets and WBC concentration also varies according to the centrifugation protocol followed which definitely has a huge impact in the expected clinical responses and the functional outcome measures.

CONSISTENCY OF PRP YIELD AS PER LITERATURE
[20] 9. The mean therapeutic dose of PRP is 1 million/cu mm of PRP in 5 ml of PRP as per literature [9], yet we are able to achieve 411*1 03 /cu mm of PRP 10. The mean PCF of 3 to 4 and PRR of 70-80% as per literature [7] are near normally reached by manual customized techniques also 11. The injection being carried out in the tendon, the volume of PRP should thus be minimal (to decrease the intratendinous pressure and to minimize pain). Hence it is necessary that PRP should also have raised platelet count invariably for producing desired output. The quantity of released growth factors could be related to the system of preparation employed [6] 12. The notification of these aspects ensures the overall quality of PRP by allowing the diversity of results to become narrowed 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 PRP in cost-effective wayfor specific clinical applications than the commercial PRP kits.

Conclusion:-
Customization of PRP preparation protocol definitely helps in standardization and hence better yield of WBC and Platelets in the final PRP Sample. The quality of the PRP contents in terms of WBC as well as Platelets yielded, improves the quality of the intervention performed and better functional outcome.
The Platelet concentration factor and Recovery Ratio of the platelets sequentially increased on refining and customizing the centrifugal acceleration forces.
Low speed centrifugation concept (LSCC) selectively enriches leukocytes, platelets and growth factors within fluid PRP. [13] A novel, custom made, economical protocol enabling us to get the procedure done in a simple and efficient manner with readily available resources, could produce PRP of comparable quality to those of a commercial PRP processing system making us to be more resilient, inclusive, cost effective and productive for a substantial better clinical outcome [8] Future Scope-Confirmatory studies will no doubt need to be completed in future. [15] Performance based standards shall be improved. Additionally, the high cost of commercially available PRP kits, hinders the liberaluse of PRP over a larger population which can be addressed in future with customised protocols of rationalisation.

Limitations
The Sample after first spin is not analyzed in my study which shall be considered for comparison in future studies. However, a manual method is susceptible to operator error, preluding to chances of bias. [12]