Original Research

Short-Term Storage of Platelet-Rich Plasma at Room Temperature Does Not Affect Growth Factor or Catabolic Cytokine Concentration

Publish date: April 13, 2018

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Statistical Analysis

Data were tested for the normal distribution to determine the appropriate statistical test. Manual and automated platelet counts were compared to each other in whole blood, L^lo PRP, and L^hi PRP samples using a paired t test. Bioactive TGF-β1 concentrations in blood, L^loPRP, and L^hiPRP, were compared using a Kruskal-Wallis one-way analysis of variance (ANOVA) with Dunn’s all-pairwise comparison. Bioactive and pro-MMP-9 concentrations measured in the retained blood or PRP samples were compared using a one-way ANOVA with Tukey’s all-pairwise comparison. Statistical analyses were performed using Statistix 9 software (Analytical Software). A P value of <0.05 was considered significant.

RESULTS

Validation of PRP

PRP, as defined by an increase in platelet concentration in PRP compared with blood, was successfully generated in all samples by both systems. There was an average 1.98 ± 0.14-fold increase in platelet concentration in L^loPRP and an average 3.06 ± 0.24-fold increase in L^hiPRP. Platelet concentration was significantly higher in L^hiPRP than in L^loPRP (P = 0.001). Compared to whole blood, WBC concentration was 0.47 ± 0.07-fold lower in L^loPRP and 1.98 ± 0.14-fold greater in L^hiPRP. Similar to platelets, WBCs were significantly greater in L^hiPRP than in L^loPRP (P = 0.02).

Bioactive TGF-β1 and MMP-9 Concentration in Blood Retained at Room Temperature

To reflect the clinical situation where blood would be drawn from a patient, but there would be a delay in processing the blood to generate PRP, blood samples were retained at room temperature for up to 4 hours prior to analysis. Neither bioactive TGF-β1 (Figure 3) nor bioactive/pro-MMP-9 concentrations (Figure 4) changed significantly over time when blood was retained at room temperature prior to centrifugation to generate PRP.

Bioactive TGF-β1 and MMP-9 Concentration in PRP Retained at Room Temperature

In order to mimic the clinical situation where PRP would be generated but might sit out prior to being administered to the patient, PRP samples were retained at room temperature for up to 4 hours prior to analysis. In these samples, bioactive TGF-β1 concentrations were not significantly different between PRP products analyzed immediately and those samples retained at room temperature for up to 4 hours (Figure 5).

Bioactive/pro-MMP-9 concentrations were also unaffected by retention at room temperature for up to 4 hours (Figure 6).

Automatic vs Manual Platelet Count

Manual platelet counts were compared to automated platelet counts to determine if a manual platelet smear analysis could be a reliable method for analyzing PRP in clinical and pre-clinical studies. There was a significant difference between the automated and manual platelet counts in blood samples (Table) (P = 0.05, N = 5) with the manual platelet count having a higher average (99.1 thou/uL) platelet concentration than automated counts. Platelet clumping was identified in 2 automated counts, which falsely decreased platelet concentration by an unknown quantity. Manual platelet counts for both L^loPRP (n = 30) and L^hiPRP (n = 30) were not different from automated platelet counts. Platelet clumping was not reported on any manual platelet counts performed on PRP samples.

Table. Platelet Concentrations of Whole Blood, L^loPRP, and L^hiPRP (N = 5)

Platelet Concentration (thou/uL)
	Automated Count		Manual Count		P Value
	Mean ± SD	Range	Mean ± SD	Range
Blood	111.8 ± 59.5	54-202	210.9 ± 59.4	144-297	0.05
L^loPRP	421.4 ± 132.8	319-620	410.1 ± 94.2	318-543	0.61
L^hiPRP	634.4 ± 88.8	517-766	635.4 ± 176.6	491-933	0.99

A paired t test was performed to compare results obtained from an automated platelet count and those obtained from a manual count.

Abbreviations: L^hi PRP, high-leukocyte platelet-rich plasma; L^lo PRP, low-leukocyte platelet-rich plasma; SD, standard deviation.

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