Effects of various centrifugation speeds and inclusion of buffy coat in PRP preparation- IJDVL study

Platelet-rich plasma (PRP) is an autologous blood preparation used in medical field because of its regenerative properties. There is a wide variation in platelet-rich plasma preparation protocols. The target platelet yield of >1 million platelets/μL in a clinic setting can be challenging to achieve. Recently a study analyzing the effect of centrifuge spin rates and inclusion of the buffy coat after the first spin on the final platelet concentration in PRP was published in the Indian Journal of Dermatology Venereology and Leprology journal.

Platelet-rich plasma can be classified into four groups according to the concentration of cellular components in it: leukocyte-poor platelet-rich plasma or pure platelet-rich plasma, leukocyte-rich platelet-rich plasma, pure platelet-rich fibrin and leukocyte and platelet-rich fibrin.5 The clinical indications for leukocyte-rich platelet-rich plasma and leukocyte-poor platelet-rich plasma in dermatology vary. Leukocyte-rich platelet-rich plasma is found effective in chronic ulcers of varied etiologies, acne scars and scar rejuvenation, while leukocyte-poor platelet-rich plasma shows good results in vitiligo and facial rejuvenation

This was a cross-sectional observational study conducted at a tertiary care center in Goa over a period of three months from December 2019 to February 2020. Seventy-five whole blood samples were obtained and divided into two groups – (1) leukocyte-rich platelet-rich plasma group and (2) leukocyte-poor platelet-rich plasma group.

Twenty milliliters of whole blood were obtained by venepuncture, out of which 10 ml was transferred into two ethylenediaminetetraacetic acid tubes. One mL was sent for complete blood count and the rest (9 ml) used for platelet-rich plasma preparation. Samples in both groups were centrifuged using the dual spin method, at one of three centrifugation speed combinations (initial "soft" spin and second "hard" spin speeds, respectively): (1) 100 g/400 g, (2) 350 g/1350 g and (3) 900 g/1800 g. Platelet, red blood cell (RBC) and white blood cell (WBC) counts in both groups were compared.

Relative centrifugal force values are used instead of rpm (revolutions per minute) for easy comparison. Relative centrifugal force is expressed as "g" and which is derived from the formula: g = (1.118×10-5) RS² where R is the radius of the rotor (in centimeters) and S is the speed of the centrifuge in revolutions per minute (rpm). g values were calculated for the respective RPM and approximated to the nearest whole number (the nearest fifty or hundred):

Variation 1 - 900/1800 rpm =104g/417g (100g/400g)

Variation 2 - 1600/3200 rpm = 330g/1329g (350g/1300g)

Variation 3- 2500/3500 rpm = 805g/1578g (900g/1800g)

Following the first spin, three layers (red blood cells (RBC), buffy coat and plasma) were formed from whole blood. For the second spin, the sample was separated and transferred into sterile plain tubes by either one of these two methods-

1. Pure platelet-rich plasma method (leukocyte-poor platelet-rich plasma): only the supernatant plasma layer was separated from the soft spin tube without disturbing the buffy coat or RBC layers, using a 2ml disposable syringe with 24G needle and subjected to a second spin. The duration of spin was ten minutes.

2. Buffy coat method (leukocyte-rich platelet-rich plasma): supernatant plasma was collected along with the whole buffy coat layer using a 2 ml disposable syringe with 24G needle and subjected to second spin. The duration of spin was ten minutes.

Patterned alopecia was the most common indication (85%) for PRP treatment followed by leg ulcers. The 100 g/400 g spin gave a high platelet yield (increase of 395.4 ± 111.1%) in the leukocyte-poor-platelet-rich plasma group, while in the leukocyte-rich platelet-rich plasma group both 100 g/400 g and 350 g/1350 g spins resulted in significantly higher yields with an increase of 691.5 ± 316.3% and 738.6 ± 193.3%, respectively. The difference between the two variations was not statistically significant (P = 0.286). The mean increase in the 800/1600 g spin was 379.8 ± 224.0%. The difference between the 800/1600 g spin (lower mean increase in platelet concentration) versus the 100/400 g and 350/1350 g variations (higher values) was statistically significant of (P = 0.000459 and 0.000165, respectively).

Platelet yields between pure-platelet-rich plasma and leukocyte-rich platelet-rich plasma groups compared

When compared to pure platelet-rich plasma (leukocyte-poor platelet-rich plasma), inclusion of the buffy coat layer for the second spin (leukocyte-rich platelet-rich plasma) resulted in 174.9%, 1222.9% and 513.7% higher platelet concentrations in the 100 g/400 g, 350 g/1350 g and 800 g/1600 g spin variations, respectively. This denotes that in the 100 g/400 g method 40–50% of platelets were in the buffy coat, whereas after the 350 g/1350 g spin nearly 90% of the platelets were in the buffy coat and only 10% in the plasma. Overall, there was no significant difference with regard to the platelet yield between (100 g/400 g and 350 g/1350 g) speeds in the buffy coat method.

Even though the buffy coat inclusion method offers higher platelet concentrations in platelet-rich plasma, it comes at the cost of higher contamination with RBCs and white blood cells (WBCs). The increase in the WBCs and RBCs was seen more in the 100 g/400 g spin as compared to other spins in the pure platelet-rich plasma samples.

This study found that inclusion of the buffy coat helped in attaining around 4–7 times rise in platelet concentrations with the highest mean value of 738.6% rise (15.316 lakh platelets/mm3) in the 350 g/1350 g spin.

In conclusion this study shows that an ideal platelet yield in platelet-rich plasma can be achieved with both the 100 g/400 g as well as the 350 g/1350 g spins while using the buffy coat (leukocyte-rich-platelet-rich plasma), and the 100 g/400 g spins method for pure platelet-rich plasma (leukocyte-poor-platelet-rich plasma) accomplishes a near-ideal platelet count along with low contamination with other cells.

Source- Muthuprabakaran K, Pai VV, Ahmad S, Shukla P. A cross-sectional analysis of the effects of various centrifugation speeds and inclusion of the buffy coat in platelet-rich plasma preparation. Indian J Dermatol Venereol Leprol 2021;87:792-9.