Abstract
Objective
To compare asinine platelet-rich plasma (PRP) manually produced by single- and double-centrifugation methods.
Methods
This was a single-center study conducted from June 19 through August 14, 2022, using 6 healthy donkeys. Whole blood (WB) was collected into sodium citrate vacutainer tubes for single-centrifugation processing and an acid-citrate-phosphate-dextrose-adenine blood bag for double-centrifugation processing to produce, respectively, PRP1 and PRP2. Platelet, WBC, and RBC concentrations and PDGF-BB and TGF-β1 activities were assessed in WB, PRP1, and PRP2.
Results
Both protocols concentrated platelets (PLTs) 1.8- to 5.2-fold, reduced WBCs 1.1- to 50.4-fold, and decreased RBCs at least 829-fold compared to WB. Platelet-rich plasma-2 yielded a higher PLT concentration and PLT enrichment factor than PRP1 but required resuspension of the pellet post second spin to maximize PLT concentration. Platelet-rich plasma-1 possessed a lower WBC concentration and greater WBC reduction factor than PRP2. Platelet-derived growth factor-BB and TGF-β1 activities were highest in PRP2 and not significantly different between PRP1 and WB. There was a weak and moderate correlation of baseline PLT concentration to that of PRP2 (r = 0.4) and PRP1 (r = 0.62), respectively; neither was statistically significant. Platelet-rich plasma-2 yielded higher PLT enrichment and growth factor activities despite greater WBC and RBC contamination than PRP1.
Conclusions
In donkeys, double centrifugation results in leukocyte-rich PRP with a higher PLT concentration compared to leukocyte-poor PRP with a lower PLT concentration yielded from single centrifugation.
Clinical Relevance
Asinine PRP can be manually prepared. While this improves cost-efficient field management of donkeys often treated under resource limitations, the optimal cellular composition, in vivo efficacy, and safety of asinine PRP warrant further investigation.
Platelet-rich plasma (PRP) is a biologic therapeutic used in human and veterinary medicine for the treatment of various orthopedic,1–5 soft tissue,6,7 dermatologic,8–10 and reproductive conditions.11–13 Its immunomodulatory effects are attributed to the supraphysiologic concentrations of platelets (PLTs) with antimicrobial peptides,3,9,14–17 growth factors,15,16 and signaling molecules15,16 that accelerate the regenerative process without major risk to the patient. The production of PRP operates by the principle of gravitational cellular filtration wherein cellular constituents become sedimented by their specific gravities through acceleration force18 or gravity. Despite the variety of existing protocols, all methods generally follow a sequence of whole-blood collection and initial centrifugation to separate erythrocytes and PLTs within plasma, with or without repeat centrifugation to further concentrate PLTs.18 In humans, double centrifugation seems to produce a higher PLT-concentrated product compared to single-centrifugation methods.18–20 This is similarly true in horses21 and dogs.22
Equine PRP production falls under commercial4,5,23 or manual noncommercial methods.10,21,23–28 The production of asinine PRP has been extrapolated from horses and can be similarly made commercially29 or manually using single30 or double31,32 centrifugation methods. The latter confers logistical and financial advantages to clinicians working with donkeys in resource-limited environments by circumventing difficulties with sourcing commercial kits as they can be made without sophisticated equipment. However, no studies have yet critically evaluated the cellular constituents of PRP produced from these noncommercial protocols.
Therefore, the objective of this study was to evaluate the effect of differential centrifugation on the composition of manually produced PRP in donkeys. The hypotheses were that manual methods could yield plasma products containing 2-to-8-fold-higher PLT concentrations than baseline in donkeys and that double centrifugation could produce PRP with a higher PLT concentration and growth factors than that of single centrifugation.
Methods
Animals
Six donkeys from the Ross University School of Veterinary Medicine (RUSVM) research herd were eligible for inclusion if they were healthy on the basis of physical examination, had a body condition score of 4 to 6 on a 7-point scale (1 [emaciated] to 7 [obese]),33 and had a normal CBC.34,35 Study animals were housed in outdoor grass paddocks under natural light with access to shade, freshly cut New Guinea grass (Megathyrsus maximus), and ad libitum water and mineral salt.
This study was carried out from June 19 through August 14, 2022, at the RUSVM. All protocols were reviewed and approved by the RUSVM IACUC (protocol No. 21.11.38).
Blood collection
Three blood collections were performed per animal, with a 2-week washout between collections. Two out of 3 manual PRP preparation methods previously validated in horses21 (double and single centrifugation) were investigated in this study; the third method from the horse study (sedimentation) was not evaluated.21 All donkeys had blood samples first collected through direct venipuncture of the jugular vein into 12 2.7-mL evacuated plastic tubes containing 3.2% sodium citrate (Vacutainer; Becton Dickinson), then into a 150-mL blood transfusion bag containing 21 mL of citrate-phosphate-dextrose solution with adenine (ACD-A; Fresenius Kabi AG). An aliquot of whole blood (WB) was also collected at every blood collection from each donkey into a 3-mL evacuated plastic EDTA tube (Vacutainer; Becton Dickinson) and submitted for automated CBC analysis (Vetscan HM5 Hematology Analyzer; Zoetis). Plasma samples from each aliquot were stored at −80 °C until batch analysis of baseline PDGF-BB and TGF-β1 activities.
Single-centrifugation processing of PRP
For the production of single-centrifugation PRP (PRP1), WB collected into citrate tubes was centrifuged using a swing-out rotor centrifuge (Centrifuge 5810R; Eppendorf) at 120 X g for 10 minutes at room temperature with the brake off (Figure 1). After centrifugation, the top third layer of plasma was considered single-centrifugation PLT-poor plasma (PPP; PPP1), with the remaining bottom two-thirds above the buffy coat considered PRP1.
Diagram demonstrating platelet-rich plasma (PRP) preparation in donkeys by single- (row 1) or double-centrifugation (row 2) processing of venous whole blood (WB) collected from 6 healthy donkeys from June 19 through August 14, 2022. For single-centrifugation PRP (A), blood collected in 2.7-mL evacuated plastic tubes containing 3.2% sodium citrate was processed by 1-step centrifugation. For double-centrifugation PRP (B), WB collected in a 150-mL blood transfusion bag containing citrate-phosphate-dextrose adenine was processed by 2-step centrifugation. PPP = Platelet-poor plasma.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.25.01.0022
Double-centrifugation processing of PRP
For the production of double-centrifugation PRP (PRP2), WB collected into a 150-mL ACD-A transfusion bag was split into 2 50-mL conical tubes (Falcon Centrifuge Tubes; Corning) for initial centrifugation (Centrifuge 5810R; Eppendorf) at 400 X g for 15 minutes at room temperature (Figure 1). Subsequently, plasma (excluding the buffy coat) was transferred into 15-mL conical tubes (Falcon Centrifuge Tubes; Corning) for a second centrifugation at 1,000 X g for 10 minutes at room temperature. The pellet was resuspended with the bottom 2.5 mL of plasma in each tube and preserved as PRP2. The supernatant above the 2.5 mL was considered double-centrifugation PPP (PPP2). All centrifugations in producing PRP2 were performed with the brake off. Resuspension was performed by gentle pipetting, taking meticulous care to avoid generating enough force to mechanically activate the PLTs within PRP2.
Evaluating the effect of pellet resuspension in the processing of PRP2
During the first blood collection of ACD-A WB, samples from 5 of 6 study donkeys were randomly selected to evaluate if pellet resuspension influenced the final PRP2 composition. Plasma, after the second centrifugation, was divided into 2 aliquots. The first aliquot was processed as described previously for PRP2 (ie, with the pellet resuspended after the second centrifugation). The second aliquot was processed such that the bottom 2.5 mL of plasma in each tube was preserved as PRP without resuspending the pellet. This was denoted as PRP2 with no pellet (PRP2.NP).
Hemogram and serum biochemistry analysis
Platelet, WBC, and RBC concentrations in the PRP and PPP of both methods were assessed by hemocytometer. For RBC and WBC counts, a 1:100 dilution of PRP or PPP was made using 0.9% NaCl solution. Samples were mixed thoroughly and loaded into the hemocytometer. Once the chambers were loaded, the sample was allowed to sediment for 10 minutes in a humidified chamber. The average number obtained from counting both chambers was used for the final statistical analysis. If the counts obtained from each side of the hemocytometer differed by > 10%, the counts were rejected, and the procedure was repeated. For PLT counting, a 1:100 dilution of PRP or PPP was made using 1% ammonium oxalate solution; samples were mixed thoroughly and allowed to rest for 5 minutes before loading the hemocytometer. Hemocytometer preparation and cell counting were performed as described above. Platelet, WBC, and RBC enrichment factors of all products were calculated as the percentage ratio between the concentration of a parameter and its baseline value.21,23
The activities of PDGF-BB and TGF-β1 were analyzed in pools of extracted PRP and PPP, and baseline single-centrifugation and double-centrifugation plasma using a sandwich ELISA technique (human PDGF-BB and TGF-β1; Quantikine; R&D Systems) after 1 freeze-thaw cycle according to the manufacturer’s instructions as previously described.23 Validation was done to assess possible matrix effects within the assay when using donkeys by spiking 6 pool samples. Plasma pools were prepared from the samples collected from the study donkeys. Spike samples were performed in duplicates by adding half a dose of known standard from the ELISA kit and half a dose of known donkey sample to a plate well. The spike recoveries (%recovery = [observed concentration/expected concentration]*100) were 99.2% and 107.1% for PDGF-BB and 106.80%, 94.76%, 95.62%, and 94.44% for TGF-β1. Thereafter, the assay was conducted according to the manufacturer’s protocol using 50 μL of plasma pools diluted to 1:20 for the PDGF kit and 1:200 for TGF-β1 with assay buffer. All samples were measured in duplicate and appropriately diluted to fit the calibration curve. The assay sensitivity was 15 pg/mL for PDGF-BB and 2.38 pg/mL for TGF-β1.
Statistical analysis
All statistical analyses were performed using RStudio R, version 4.2.3 (R Core Team). The normality of data was assessed by the Shapiro-Wilk test (PLT concentration, WBC concentration, and RBC concentration; their enrichment factors; and PDGF-BB and TGF-β1 activities). Nonparametric data was further evaluated by the Friedman test followed by the Dunn multiple comparison test (function “friedman_test” in the R “agricolae” package).36 For all analyses, the donkey was accounted for as a random effect, with groups (WB, PRP1, PPP1, PRP2, and PPP2) and samplings (first, second, and third) as fixed effects. The correlation between PLT concentration in WB and PRP for each centrifugation method was tested using the Spearman rank correlation. A strong correlation coefficient was defined as r > 0.7, moderate as 0.5 ≥ r ≤ 0.7, and weak as r < 0.5. Significance was set at P ≤ .05.
Results
Animals
Three johns and 3 jennies aged 4 to 8 years old and weighing 110 to 134 kg met the inclusion criteria. Complete blood count results of the enrolled donkeys are featured in Table 1.
Selected baseline hemogram values (platelet, WBC, and RBC concentrations) from 6 healthy donkeys from June 19 through August 14, 2022.34
Donkeys | Platelets (X 103/µL) | WBCs (X 103/µL) | RBCs (X 106/µL) |
---|---|---|---|
1 | 139 | 9.4 | 5.7 |
2 | 140 | 10.4 | 5.6 |
3 | 156 | 7.0 | 4.9 |
4 | 201 | 7.1 | 5.3 |
5 | 162 | 7.0 | 5.4 |
6 | 165 | 8.5 | 5.5 |
Platelet-rich plasma-2 processed with and without pellet resuspension
Platelet-rich plasma-2 yielded higher PLT concentration (mean ± SD X 103/µL; Figure 2) compared to WB (PRP2, 739.5 ± 70.0; WB, 129.4 ± 34.7; P < .05), whereas PRP2.NP produced the lowest PLT concentration (59.2 ± 17.6) compared to both PRP and WB (P < .05). White blood cell concentration (mean ± SD X 103/µL) was similar between PRP2 and WB (PRP2, 8.4 ± 4.8; WB, 9.1 ± 1.4; P > .05) and lowest in PRP2.NP of all groups (1.3 ± 0.5; P < .05). Red blood cell concentration (mean ± SD X 109/µL) was reduced in both PRP groups (PRP2, 0.6 ± 0.3; PRP2.NP, 0.1 ± 0.1) compared to WB (605 ± 54; P < .001).
Concentrations of platelets (A) and leukocytes (B) in WB, PRP produced by double centrifugation with pellet resuspension between centrifugations (PRP2), and PRP produced by double centrifugation without pellet resuspension between centrifugations (PRP2.NP) from 5 of 6 healthy donkeys (as described in Figure 1). Data are presented as the mean concentration ± SD. For each plot, the horizontal line in the box represents the median; the upper and lower limits of the box represent the 25th and 75th percentiles, respectively; the whiskers represent the range of the data; the red diamond represents the mean; each circle represents an individual data point; and the shading within the violin shape represents the distribution of data, with wider shapes indicating an increased frequency of data points in that region and the opposite for narrower shapes. Different letters indicate statistical significance (P < .05) between products.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.25.01.0022
Characteristics of PRP1, PRP2, PPP1, and PPP2
Platelet concentration was increased in PRP2 (728.0 ± 151.8) and PRP1 (235.7 ± 53.1) compared to WB (132.3 ± 29.0; P < .05; Figure 3). Similarly, the PLT enrichment of both protocols was significantly higher than baseline (PRP2, 5.5 ± 1.8; PRP1, 1.8 ± 0.71; P < .05). However, PRP2 yielded significantly higher PLT concentration and PLT enrichment than PRP1 (P < .05). Baseline PLT concentration was moderately correlated with the PLT concentration in PRP1 (r = 0.62; P > .05) and weakly correlated with PLT concentration in PRP2 (r = 0.42; P > .05). Irrespective of centrifugation technique, PLT concentrations of PPP1 and PPP2 were not statistically different to each other or with WB (PPP1, 161.1 ± 36.6; PPP2, 135.9 ± 30.5; P > .05).
Concentrations of platelets (A) and WBCs (B) and their respective enrichment factors (C and D) in WB, PRP2, PPP produced by double centrifugation (PPP2), PRP produced by single centrifugation (PRP1), and PPP produced by single centrifugation (PPP1) from 6 healthy donkeys (as described in Figure 1). Data are presented as the mean concentration ± SD. For each plot, the horizontal line in the box represents the median; the upper and lower limits of the box represent the 25th and 75th percentiles, respectively; the whiskers represent the range of the data; the red diamond represents the mean; each circle represents an individual data point; and the shading within the violin shape represents the distribution of data, with wider shapes indicating an increased frequency of data points in that region and the opposite for narrower shapes. The dashed lines in the graphs depicting platelet (C) and WBC enrichment (D) represent the baseline platelet and WBC concentrations in WB that were used to calculate platelet and WBC enrichment, respectively. Different letters indicate statistical significance (P < .05) between products.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.25.01.0022
White blood cell concentration was significantly decreased in all samples compared to WB (8.9 ± 1.0) except for PRP2 (7.8 ± 5.4; P < .05; Figure 3). The mean WBC concentration in PRP2 was lower, but not statistically different, than that of WB (P > .05). Nonetheless, there was a 14% reduction in WBC enrichment in PRP2 (0.86 ± 0.54) compared to baseline (P < .05). All other samples also resulted in significantly decreased WBC enrichment compared to WB (PRP1, 1.8 ± 1.0; PPP1, 0.8 ± 0.6; PPP2, 0.1 ± 0.3; P < .05).
Similarly, RBC concentration was significantly decreased in PRP- and PPP-derived products from both methodologies compared to WB (P < .001). Platelet concentration and WBC concentration in WB were poorly correlated to the concentrations in PRP1 (PLT concentration: r = −0.06, P = .8; WBC concentration: r = 0.38, P = .1) and PRP2 (PLT concentration: r = 0.04, P = .9; WBC concentration: r = 0.33, P = .2).
Unlike PRP1, both PDGF-BB (mean ± SD X 103 pg/mL) and TGF-β1 (mean ± SD X 103 pg/mL) activities were significantly increased in PRP2 compared to WB (PDGF-BB 1.7 ± 0.5; TGF-β1 5.9 ± 1.1; P < .05; Figure 4). The PLT-derived growth factor-BB activities of PRP1 (2.1 ± 0.6) and PRP2 (12. 0 ± 3.8) were significantly different from PPP1 (1.4 ± 0.5) and PPP2 (0.4 ± 0.2), respectively. The transforming growth factor-β1 activity of PRP2 (13.2 ± 3.4) was significantly higher than PPP2 (6.3 ± 1.1), but this was not similarly different between PRP1 (6.1 ± 2.5) and PPP1 (5.8 ± 1.8; P < .05). The activities of PDGF-BB and TGF-β1 were similar across all PPP samples and WB (P > .05).
Activities of PDGF-BB (A) and TGF-β1 (B) in WB, PRP2, PPP2, PRP1, and PPP1 from 6 healthy donkeys (as described in Figure 1). Data are presented as the mean concentration ± SD. For each plot, the horizontal line in the box represents the median; the upper and lower limits of the box represent the 25th and 75th percentiles, respectively; the whiskers represent the range of the data; the red diamond represents the mean; each circle represents an individual data point; and the shading within the violin shape represents the distribution of data, with wider shapes indicating an increased frequency of data points in that region and the opposite for narrower shapes. Different letters indicate statistical significance (P < .05) between products.
Citation: American Journal of Veterinary Research 86, 6; 10.2460/ajvr.25.01.0022
Discussion
The results of this study demonstrate that asinine PRP can be prepared using the previously validated methodologies described in horses.21 Interspecies comparison shows that the PLT enrichment of manually produced asinine PRP is similar to that of manually produced equine PRP using a nearly identical double-centrifugation technique.21 However, the leukocyte composition was different than that previously reported in horses21 (Supplementary Table S1).
Aligning with existing literature in other species, double centrifugation also produced asinine PRP (ie, PRP2) with a higher PLT concentration compared to single centrifugation (ie, PRP; Supplementary Table S1). Further, the higher PLT concentration in asinine PRP2 demonstrated the highest activities of both PDGF-BB and TGF-β1 among all PLT products. In the methodology employed in this study, PLT yield in PRP2 could only be maximized with pellet resuspension after the last centrifugation step, which also increased the WBC contamination in PRP2 compared to PRP1. To the best of the authors’ knowledge, this is the first study in donkeys to compare different manual methods of PRP preparation within the same study.
Confounding critical analysis of PRP characteristics is the lack of consensus regarding the optimal cellular composition of PRP in human or veterinary medicine.37,38 Recommendations are complicated by the likelihood that therapeutic concentrations are most certainly specific to the condition and/or tissue being treated.38 In humans, a PLT concentration of 1,000,0000/µL is considered the definition of therapeutic PRP,18 with concentrations of 0.5 X 1011 PLTs/U acting as the standard in human transfusion medicine.39 In horses, equine PLT concentrate produced by multiple sequential centrifugations (ie, greater than 2) yielded a concentration of > 550 X 103 PLTs/μL.40 The PLT concentration of PRP2 in this study exceeded those criteria. These results seem to support that multiple centrifugation steps increase the PLT enrichment in PLT-concentrate products and suggest that double centrifugation is superior to single centrifugation when aiming to produce asinine PRP with a greater PLT concentration. The weak correlation between the PLT concentration of WB and PRP2 in this and other horse studies40 also lends support to this hypothesis. While the absolute PLT concentration of PRP2 did not exceed the human benchmark of 109/mL,41 the PLT concentration yielded in PRP2 was greater than 4 times the PLT concentration baseline concentration in asinine WB and had a similar PLT enrichment as that of equine PRP produced via double centrifugation (Supplementary Table S1).
The PPP consistently yielded low PLT concentration regardless of the number of centrifugations in the present study. This aligned with the results of low PDGF-BB and TGF-β1 activities in PPP1 and PPP2 as PLT-derived growth factor activity is proportionally related to the PLT concentration.42,43 The major effector molecules in PRP are attributed to growth factors highly concentrated in PLT granules.44–46 This is the first study to measure both PDBF-BB and TGF-β1 in asinine PRP; only 1 previous study30in donkeys measured growth factor activity (TGF-β1) in the final PRP produced. Based on the results of the present study, there is a positive association between PLT concentration and growth factor activities, indicating that PRP1 and PRP2 are viable products with therapeutic potential. Ultimately, it must be kept in mind that ideal PLT concentration for therapeutic efficacy in donkeys remains unknown and requires further investigation.
Unlike horses, enrichment of WBCs in asinine PRP after double centrifugation was significantly higher than after single centrifugation (Supplementary Table S1). Based on the results of the first experiment in which the effect of pellet resuspension on PLT concentrations was assessed, the authors suspected that an inherent species-specific difference in the plasma composition of donkeys could affect the final composition of PRP after repeat centrifugation. This influenced the decision to evaluate the effect of PLT resuspension on PRP2 and PRP2.NP. In horses, resuspension of the pellet was not necessary between sequential centrifugation to obtain PLTs during the double-centrifugation method,21 whereas not resuspending the pellet in this study resulted in significantly less PLT yield. In fact, the absolute concentration of PLTs in PRP2.NP was less than PRP1. Although the identification and comparison of specific plasma constituents between horses and donkeys are beyond the scope of this study, it is well known that donkeys have naturally higher reference ranges for serum triglycerides.47 The authors believe that this provides a reasonable theoretical basis to conjecture that inherent differences may exist between asinine and equine plasma density, which interfere with the ability to sediment WBCs from PLTs in donkey plasma.
Variability in handling between double-centrifugation and single-centrifugation protocols as inherent to manual PRP production methods may have also influenced the increased WBC presence in PRP2 compared to PRP1. All procedures performed were done so by the same trained personnel to maximize standardization, but it remains possible that WBC concentration and WBC enrichment could have been reduced if commercial kits had been used instead. Arguably, both study protocols involved the careful removal of plasma above the buffy coat to optimize PLT yield and minimize WBC contamination in the final PRP product and should have produced leukocyte-poor PRP regardless of number of centrifugations. In PRP1, the bottom two-thirds of the centrifuged plasma above the buffy coat was removed and considered PRP. In PRP2, the plasma after the first spin above the buffy coat was removed to undergo a second spin. This plasma should have already contained minimal WBCs. After the second spin, it would be expected for heavier WBCs to be sedimented out into the pellet without any influence on PLT concentration in the resulting PRP above the pellet. However, as is shown in Figure 2, the significant difference between PLT and WBC concentrations in PRP2 (which incorporated pellet resuspension) versus PRP2.NP (which did not incorporate pellet resuspension) strongly suggest that human error intrinsic to manual production methodologies alone is unlikely to be the only factor affecting the final composition of PRP2. This is the rationale for the authors’ previous supposition that there may be unique, unidentified elements within donkey plasma that cause both PLTs and WBCs to be pulled concurrently into the pellet during the second centrifugation. Further refinement of techniques to enrich PLT concentration while decreasing WBC concentration in the manual production of PRP may be desired depending on the intended use of PRP.
The increased WBC contamination seen in PRP2 may limit its clinical utility. However, the presence and influence of non-PLT cellular components on the efficacy of PRP remains controversial in the literature.42,46,48–50 Opinions are divided between studies46,49 suggesting that increased secretion of proinflammatory cytokines in leukocyte-rich PRP (LR-PRP) could damage treated tissue, those proposing that there may be a cooperative anti-inflammatory effect between WBCs and PLTs in PRP,50 and others suggesting that LR-PRP efficacy may be affected by when it is used during the timeline of healing48,51 and the type of injury52 being treated. Leukocytes are also thought to contribute antimicrobial properties to PRP,52–54 although there is debate about whether this is directly related to WBCs in PRP given that PLTs also have antimicrobial properties.3,9,14–17 In equine tendon explant models, leukocyte presence was shown to correlate with increased expression of catabolic genes,5 and a high absolute WBC concentration contributed to significantly increased expression of proinflammatory cytokines.46 Conversely, in a randomized clinical trial involving race horses with proximal sesamoiditis and suspensory ligament desmitis, LR-PRP–treated horses were more likely to start racing than the placebo group.55
Meta-analyses of human clinical trials also seem to show positive outcomes in treating tendinopathies with LR-PRP.56,57 Ex vivo studies58 using torn human rotator cuffs showed increased tenocyte proliferation in LR-PRP compared to leukocyte-poor PRP treatment groups. In rats, LR-PRP was shown to accelerate healing, hasten the inflammatory response, and stimulate more prominent angiogenesis in a pressure ulcer model compared to leukocyte-poor PRP.59 Unsurprisingly, the results of ex vivo studies, particularly those using healthy tissue, do not fully capture the complex biological environment and interplay of cells and immune mediators involved in tissue healing. Therefore, results may not always be clinically applicable. Further in vivo studies are necessary to determine the clinical efficacy of manually produced PRP and the effect of LR-PRP and leukocyte-poor PRP in the treatment of a variety of clinical conditions in donkeys.
Pertinent to this study, a WBC differential was not available on the final PRP products as WBC concentration was determine manually by hemocytometer, and not an automated hematology analyzer, in all postcentrifugation samples. Without more information about the type of WBCs present in PRP2, the inflammatory potential of this product remains unknown given that different WBCs can exert proinflammatory or anti-inflammatory effects. The lack of a differential cell count also makes it difficult to determine to what extent the growth factor activity detected in PRP2 was derived from WBCs considering that TGF-β1 can be produced from multiple lineages.60
Other limitations of this study not yet addressed include the lack of randomization in the sequence of blood collections, which may potentially lead to an overestimation of the effect of double centrifugation as the superior method for PLT and growth factor enrichment (type I error). Additionally, while this study successfully characterizes the PLT concentration obtained through 2 manual PRP processing techniques in donkeys, it does not provide any data on the clinical efficacy or safety of PRP2 or PRP1 preparations. The decision to manually resuspend the pellet via gentle aspiration was elected to mimic the most practical manner in which general practitioners would most likely perform pellet resuspension, but it cannot be ruled out that this method may have led to some mechanical activation of PLTs and subsequently increased the growth factor activity detected in PRP2. Furthermore, although the PRP production methods described are more accessible to general practitioners, it still necessitates large benchtop centrifuges, which may be impractical for veterinarians operating in strictly ambulatory or field settings.
In conclusion, manual methods of producing PRP in horses can be used to produce PRP in donkeys. Much like in horses and other species, sequential centrifugation in the production of donkey PRP increases PLT enrichment in the final product. In the study herein, double-centrifugation processing resulted in greater PLT enrichment compared to single-centrifugation processing. This was supported by higher growth factor enrichment of both PDGF-BB and TGF-β1. However, PRP2 had significantly greater leukocyte contamination than PRP1. The clinical implication of PRP with a richer WBC concentration, and whether this characteristic impacts its efficacy, warrants further investigation.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors would like to thank the Animal Resources team for providing husbandry care for the study animals.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the composition of this manuscript.
Funding
Financial support was provided by the Ross University Center for Integrative Mammalian Research (No. 42011-2022).
ORCID
C. Xue https://orcid.org/0000-0003-2756-7835
L. G. T. M. Segabinazzi https://orcid.org/0000-0001-7526-7760
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