The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months.
Abstract: Platelet-rich plasma (PRP) is a therapeutic biologic that is used for treatment of musculoskeletal pathologies in equine athletes. Due to the expense of PRP kits, and the volumes obtained, freezing aliquots for future dosing is common. Aliquots of PRP are also commonly frozen for later analysis of growth factor concentrations in in vitro research. A variety of freezing methods are used and storage duration until analysis is often not reported. The optimal frozen storage conditions and duration to maintain concentrations of commonly measured growth factors and enzymes in PRP are unknown. Our objectives were two-fold. First, to determine the effect of a single freeze-thaw cycle on PRP protein concentrations and establish their baseline levels. Second, to evaluate the effect of storage in -20 °C automatic defrost freezer, - 20 °C manual defrost freezer, - 80 °C manual defrost freezer, and liquid nitrogen for 1, 3, and 6 months on PRP protein concentrations, compared to the established baseline concentrations. Results: Fold-change between fresh activated and snap frozen PRP were analyzed using paired t-test. A snap frozen-thaw cycle resulted in increased MMP-9 (p = 0.0021), and a small significant decrease in TGF-β1 (p = 0.0162), while IGF-1 and PDGF-BB were unchanged compared to fresh activated PRP. Fold-change over time within storage method were analyzed using repeated measures ANOVA and Tukey post-hoc test. IGF-1 decreased in all conditions (p < 0.0001). At all time-points at -20 °C (p < 0.0001), and at 3 and 6 months at -80 °C (p < 0.0070), PDGF-BB decreased. TGF- β1 was unchanged or increased after 6 months (p < 0.0085). MMP-9 decreased at 3-months at -20 °C, and at all times at -80 °C and in liquid nitrogen compared to snap frozen (p < 0.0001). Conclusions: The protein profile of equine frozen-stored PRP differs from fresh PRP. For clinical applications equine PRP can be stored at -80 °C for 1 month or in liquid nitrogen for 6 months to maintain PDGF-BB and TGF-β1 concentration, but IGF-1 concentrations will be reduced. The storage temperature and duration should be reported in studies measuring protein concentrations in PRP. To accurately measure IGF-1 concentrations, PRP samples should be analyzed immediately.
Publication Date: 2019-08-14 PubMed ID: 31412868PubMed Central: PMC6694589DOI: 10.1186/s12917-019-2040-4Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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This research article investigates how different freezing conditions and storage durations affect the concentration of certain proteins in equine platelet-rich plasma, a treatment used for musculoskeletal conditions in horses. The study suggests optimal storage conditions to retain the potency of the treatment.
Overview
- The researchers aimed to understand the impacts of differing freezing conditions and durations on the concentrations of proteins in equine platelet-rich plasma (PRP).
- PRP is a biologic therapy used to treat musculoskeletal conditions in horses. Trade practices include freezing aliquots of PRP for future use or analysis, despite limited understanding of optimal storage conditions.
Objectives
- The study set out with two key objectives:
1. To ascertain the influence of a single freeze-thaw cycle on PRP protein concentrations and deduce their baseline levels.
2. To analyze the impact of storage under different conditions -20 °C automatic defrost freezer, - 20 °C manual defrost freezer, - 80 °C manual defrost freezer, and liquid nitrogen for 1, 3, and 6 months–on PRP protein concentrations.
Results
- The study observed varied influences on different proteins after a snap frozen-thaw cycle.
- MMP-9 increased, while TGF-β1 decreased insignificantly; IGF-1 and PDGF-BB remained unchanged compared to fresh PRP.
- Over time, fluctuations in protein concentration were observed, based on the storage method.
- All conditions led to a decrease in IGF-1.
- At all time points at -20 °C and at 3 and 6 months at -80 °C, PDGF-BB decreased.
- TGF- β1 remained unchanged or increased after 6 months.
- MMP-9 dropped at 3 months-at -20 °C and at all times -80 °C and liquid nitrogen storage.
Conclusions
- Researchers concluded that the proteins in equine frozen-stored PRP differ from fresh PRP.
- For clinical applications, equine PRP can be stored at -80 °C for 1 month or in liquid nitrogen for 6 months to maintain PDGF-BB and TGF-β1 concentrations, but this will lower IGF-1 concentrations.
- Future studies reporting on PRP protein concentrations should indicate storage temperature and duration.
- To accurately measure the IGF-1 protein, PRP samples should be immediately analyzed.
Cite This Article
APA
McClain AK, McCarrel TM.
(2019).
The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months.
BMC Vet Res, 15(1), 292.
https://doi.org/10.1186/s12917-019-2040-4 Publication
Researcher Affiliations
- Department of Large Animal Clinical Sciences, University of Florida College of Veterinary Medicine, 2015 SW 16th Avenue, Gainesville, FL, 32610, USA.
- Department of Large Animal Clinical Sciences, University of Florida College of Veterinary Medicine, 2015 SW 16th Avenue, Gainesville, FL, 32610, USA. tmccarrel@ufl.edu.
MeSH Terms
- Animals
- Enzymes / chemistry
- Enzymes / metabolism
- Freezing
- Horses
- Intercellular Signaling Peptides and Proteins / chemistry
- Platelet-Rich Plasma / chemistry
- Time Factors
Grant Funding
- N/A / Department of Large Animal Clinical Sciences, University of Florida
Conflict of Interest Statement
The authors declare that they have no competing interests.
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