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

References

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When performing clinical or research studies using PRP, it is important to report the contents of the PRP delivered to the patient. By documenting the cellular content of the PRP delivered to the patient, the common questions of optimal platelet dose and the importance of leukocytes in PRP can begin to be answered. There are some known factors that contribute to PRP variability, such as patient biology and operator technique, but there are many other unknown factors. In some instances, there is a failure to generate PRP, defined as a lower platelet count in the PRP preparation than in the starting blood sample.^13,14 To measure the platelet and cellular contents of the starting blood and PRP, samples can be submitted to a clinical pathology laboratory for a complete blood count, which adds cost to the patient above the typically unreimbursed cost of the PRP injection itself. An alternative method for measuring platelet concentrations is the use of direct smear analysis on glass slides. The use of direct smears to measure platelet concentration is well validated for blood,^20,21 but the use of direct smears of PRP for determining platelet concentrations has not been previously validated. The use of manual platelet counts would provide an alternative to automated platelet counting for clinical and preclinical research studies to characterize the type of PRP administered to the patient.

The primary aim of this study was to determine if retention of blood or PRP at room temperature for various time intervals had an effect on final growth factor or catabolic cytokine concentration. Bioactive transforming growth factor-β1 (TGF-β1) and matrix metalloproteinase-9 (MMP-9) were measured as representatives of growth factors and catabolic cytokines, respectively. The secondary aim was to identify if manual platelet counts were an accurate reflection of automated counts. The outcomes of these experiments should provide immediately relevant information for the clinical application of PRP.

MATERIALS AND METHODS

Blood Collection and Generation of PRP

Under Institutional Review Board approval, blood (105 mL) was collected from healthy human volunteers (N = 5) into a syringe containing acid citrate dextrose anticoagulant to a final concentration of 10% acid citrate dextrose. Three 15-mL aliquots of blood were used to generate L^lo PRP (Autologous Conditioned Plasma Double Syringe, Arthrex) and three 20-mL aliquots were used to generate L^hi PRP (SmartPReP 2, Harvest Technologies) (Figure 1).

To test the effect of a delay in PRP generation, blood samples from each L^lo PRP and L^hi PRP group were processed into PRP either immediately or after being retained at room temperature for 2 or 4 hours. Aliquots of the resulting PRP were frozen at −80°C for future analyses. To test the effects of retaining PRP at room temperature, PRP was either frozen immediately after it was generated or kept at room temperature for 1, 2, or 4 hours prior to being frozen. All samples were centrifuged at 12,000 g for 15 minutes prior to storage to remove cells and cellular debris, and none of the PRP samples were buffered or activated after processing.

Automated and Manual Counts

Automated complete blood counts were performed by a board certified clinical pathologist in the clinical pathology department of Cornell University on all blood, L^lo PRP, and L^hi PRP samples. A manual platelet count, using a modified Giemsa stain,²² was performed on smears of all blood and PRP samples (Video). Slides were scanned at 10x magnification to identify an area where many red blood cells were present while maintaining a clear field of view (Figure 2A). The magnification was then increased to 100x using oil immersion, and the total number of platelets was counted in 10 fields of view (Figure 2B).

The average number of platelets per high-powered field was calculated and multiplied by 15 to determine total platelet concentration (thou/uL) for each sample.²² These data were used to determine the correlation between manual vs automated platelet counts.

Growth Factor and Catabolic Cytokine Measurements

Blood and PRP samples were thawed for ELISA (enzyme-linked immunosorbent assay) analysis. TGF-β1 concentration was determined using the TGF-β1 Emax ImmunoAssay System (Promega Corporation), which measures biologically active TGF-β1. We chose TGF-β1 because it is commonly measured in PRP studies as an anabolic cytokine with multiple effects on tissue healing. The functions of TGF-β1 include stimulation of undifferentiated mesenchymal cell proliferation; regulation of endothelial, fibroblast, and osteoblast mitogenesis; coordination of collagen synthesis; promotion of endothelial chemotaxis and angiogenesis; activation of extracellular matrix synthesis in cartilage; and reduction of the catabolic activity of interluekin-1 and MMPs.^23-25 In addition, TGF-β1 concentration strongly correlates with platelet concentration.²⁶ MMP-9 concentration was determined using the MMP-9 Biotrak Activity Assay (GE Healthcare Biosciences) which measures both active and pro- forms of MMP-9. In PRP, MMP-9 was measured as an indicator of white blood cell (WBC) concentration.²⁶ A catabolic cytokine capable of degrading collagen,^13,27 MMP-9 has been linked to poor healing.²⁸ For both assays, samples were measured in duplicate using a multiple detection plate reader (Tecan Safire).

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