Blood Collection
This study was conducted after being approved by the Institutional Ethical Committee. We collected blood from 40 healthy volunteers who signed informed consent for participation in the study without any comorbid illness or history of any medications in the past 3 months. We collected blood from the donors following sterile aseptic precautions and used Citrate-Phosphate-Dextrose-Adenine (CPD1) anticoagulant solution at the ratio of 1.5 ml per 10 ml of blood. We used 20 samples as the standardization cohort and 20 samples as the validation cohort of the standardized protocol. We used 15 ml centrifugation tubes Spinwin TM (Tarsons, India) or 5 ml blood collection tubes BioPro (Alchem Diagnostics, India) based on the protocol analyzed. Each protocol investigated was run twice in all the samples to avoid reporting and sampling bias. Average of the values were taken for analysis.
First spin
To standardize the protocol for the first spin 10 ml of whole blood from 20 standardization cohort samples were centrifuged at incremental acceleration rates ranging from 50 to 1000xg (50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, and 1000xg) for 10 minutes in a laboratory centrifuge R-8C (REMI, India) with swing-out 16-well R-81 rotor head (REMI, India) with rotor radius of 150 mm. To make the protocol applicable to clinical practice we performed the centrifugation at room temperature (22° C) and the centrifuges were not refrigerated. Following the first spin, the whole blood is separated into the RBC layer at the bottom, buffy coat in the middle, and plasma layer at the top. We collected the upper plasma layer without disturbing the middle/bottom layers using a pipette. Depending on the centrifugal force utilized the volume of the upper layer ranged from 2–6 ml. The sample was then thoroughly mixed and blood cell counting was performed using cell counter XP-300™ (Sysmex America, Inc.). With the minimal centrifugal acceleration giving maximum platelet recovery, we titrated the time ranging from 5–30 minutes (5, 10, 15, 20, 25, and 30 minutes) to investigate the effect of time on the recovery efficacy of centrifugation. Based on the results the final first spin centrifugation protocol was finalized.
Second Spin
The whole blood was initially centrifuged as per the identified ideal centrifugation protocol for the first spin to give 5 ml of the plasma. We performed second centrifugation in them at various centrifugal accelerations ranging from 200 to 2000xg (200, 400, 800, 1200, 1600, and 2000xg) for 10 minutes. The level of plasma to be discarded as platelet-poor plasma (PPP) was identified by performing blood cell counting of the upper, middle, and lower 1/3rd of the final plasma obtained. The PRP thus obtained by discarding the PPP was analyzed for its cellular constituents. With the minimal centrifugal acceleration giving maximum platelet recovery, we titrated the time ranging from 5–30 minutes (5, 10, 15, 20, 25, and 30 minutes) to investigate the effect of time of centrifugation in the second spin. To investigate the effect of the volume of the whole blood utilized we ran the protocol with minimal centrifugal acceleration giving maximum platelet recovery in both the first and second spin for 5, 10, and 15 ml of whole blood volume.
PRP Characterisation
The final PRP obtained was characterized by measuring the platelet concentration gradient at various levels of the final product obtained after the second spin, cell composition of the final product, and analyzing the integrity of the platelets.
First Spin Analysis
After the first spin, the concentration of platelets and WBC was measured to calculate the recovery efficiencies of the plasma (E pl), platelets (E pt), and WBC (E WBC) along with the platelet concentration factor (Fc pt). These values were calculated using the following formulae
$$\text{E} \text{p}\text{l}=\frac{Volume of upper layer}{Total volume of blood \times (1-hematocrit)}\times 100$$
$$\text{E} \text{p}\text{t}=\frac{Number of Platelets in upper layer}{Number of Platelets in whole blood}\times 100$$
$$\text{E} \text{w}\text{b}\text{c}=\frac{Number of WBC in upper layer}{Number of WBC in whole blood}\times 100$$
$$\text{F}\text{c} \text{p}\text{t}=\frac{Concentration of Platelets in upper layer}{ Concentration of Platelets in whole blood}\times 100$$
Second Spin Analysis
After the second spin, the cellular concentration of the upper, middle, and lower 1/3rd of the plasma is analyzed to identify the concentration gradient. The recovery efficiencies and the concentration factor of the platelets in the layer of the plasma were calculated using the following formulae
$$\text{E}\text{p}\text{t} \left(\text{P}\right)=\frac{Number of Platelets in plasma layer analyzed}{ Number of Platelets in upper layer}\times 100$$
$$\text{F}\text{c} \text{p}\text{t} \left(\text{P}\right)=\frac{Concentration of Platelets in plasma layer analyzed}{ Concentration of Platelets in upper layer}\times 100$$
Platelet Concentration Gradient
After the second spin, the volume of plasma was divided into upper, middle, and lower 1/3rd and analyzed for cellular components to estimate the demarcation of PPP and PRP in the final plasma product obtained. Before cell counting the separated plasma layer was mixed by inversion manually for 30 seconds to minimize the gradient effect upon cell counting.
Platelet Integrity
The effect of the centrifugal acceleration on the integrity of the platelets was assessed in the final PRP obtained after the second spin by measuring the lactate dehydrogenase (LDH) levels and comparing it to the serum baseline levels. This indirectly measures the integrity of the platelet membranes and any damage to the platelet would result in a rise of the LDH levels significantly compared to the serum baseline measured beforehand.
Protocol Validation
In all the cases, the analysis was repeated twice to prevent the reporting bias and the final protocol arrived was run on common bench-top laboratory centrifugation machines such as Medico Plus (REMI, India) and R-4C (REMI, India) to establish the validity of the PRP preparation protocol ascertained.
We used descriptive statistics to describe the results of the parameters analyzed. We presented the continuous variables with mean and standard deviation. Variables were tested for distribution normalcy using the Kolmogorov-Smirnov test and we found a normal distribution of all the variables analyzed (p > 0.30 for all variables). We used paired t-test to analyze the significance of the increase in the levels of LDH compared to the baseline. We used IBM SPSS Version 25 (Chicago, IL, USA) for statistical analysis. We considered a p value less than 0.05 as significant.