This research describes a simple centrifugation manual (tube method) protocol to obtain PC from feline blood, thereby concentrating the growth factors such as TGF-β1 and PDGF-BB for experimental or clinical application in this species. The protocol described here presents the advantage that the PC is easily obtained with one centrifugation step with a small volume of blood. This last situation is important in feline practice because the volume of blood required to obtain PC for clinical application could be a limiting factor, especially in pediatric patients. To note, both PC obtained in this study could be classified as P-PRP.
We have not found any published studies about preparation of PC (either P-PRP or L-PRP) for clinical use in cats for regenerative medicine purposes. However, we did find information regarding manual methods for concentrating feline platelets for evaluating in vitro the effect of aggregating and anti-platelet substances in this species. In those studies, sodium citrate 3.8% was used as an anticoagulant. The studies included different centrifugation protocols ranging between 150–200 g and times of centrifugation ranging from 10–20 min
[23–29]. These studies did not present data on the number of platelets concentrated or other hematological information with respect to the characteristics of those PC. For this reason, this study presents novel information about feline hematology with potential applications of PC for regenerative medicine purposes in cats.
The cellular characteristics obtained in both PC differed only in the highest concentration of LYM found in PC-A. This finding could suggest that each PC could display different biological effects mediated by this kind of cell when used clinically. This assumption can suggest a difference in clinical application characteristics of each portion due to the important regulatory effect of leukocytes in the healing process
. Particularly, lymphocytes are one major source of granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, interleukin-1 and tumor necrosis factor alpha. These proteins have functions related to wound healing because they increase the activity of neutrophils and monocytes and promote the proliferation of keratinocytes and fibroblasts. All these actions are important in the inflammatory phase of wound healing
. However, this suggested mechanism is only an assumption, and additional experimental work is necessary to evaluate this hypothesis.
The platelet collection efficiency was low (26.16% for PC-A and 24.75% for PC-B) in this study. This low efficiency is one of the main characteristics of manual methods (tube) to concentrate platelets in humans
 and horses
. However, no other published results have been found for cats to compare with these results. The platelet collection efficiency obtained in this study could be sufficient (in terms of the concentration of platelets) to produce biological effects because the high concentrations of platelets could suppress cell viability and proliferation
. This concept is still controversial and should be the subject of future studies in cats. One limitation, with the platelet count of this study was that blood smears were not made to ensure no platelet clumping, this could be a potential limitation because clumping would influence the platelet counts.
The MPV represents the average size of the platelets, and PDW is an indicator of variation in the size of the platelets. The MPV and PDW values for automated hematology instruments would be increased during platelet activation
. The MPV and PDW values were lower in whole blood that in either PC. However, these platelet activation related parameters remained in a normal rank in both PC
[36, 37]. These values would indicate that the methodology used to obtain the PC in cats did not produce platelet activation. This concept is important if we consider that an effective method for concentrating platelets should first focus on obtaining functional and non-activated platelets instead of concentrating a large number of platelets
. In the light of procoagulant properties of feline platelets, this statement must be interpreted with caution and could be a limitation of this study.
Autologous platelet concentrate preparation involves a series of centrifugation and separation cycles for concentrating the platelets without inducing premature activation. The size and weight of the blood cells and the relative forces (g) and time of centrifugation are the factors that determine the cellular and molecular characteristics of a PC. Studies in dogs
 and humans
 describe protocols with two rounds of centrifugation. One of these episodes is always greater than 240 g, and one of the centrifugation times is greater than 10 minutes (except for horses). In the case of cats, the PLT can be concentrated by a soft spin (85 g) and short time (6 minutes). This methodological difference between species to obtain PC may be due to the morphological characteristics of cat platelets, such as higher diameter (2–6 μm)
 and mean platelet volume (8.6-14.1 fL)
As described in a study in horses
, samples were kept in incubation at room temperature (20–22°C) for two hours after activation. In our case (incubation at 37°C), within two hours after activation, we did not yet have complete formation of a fibrin clot in most of the PC samples. For this reason, the cat samples were left in incubation for another hour (three hour, total incubation time). This difference in time required for the formation of fibrin clots can be due additionally to the peculiar morphology of the platelets of both species and to the cellular count of the PC and whole blood of each species. The platelet counts in whole blood and PC in horses
, are lower than those found in the cats in the present study; in particular, the PC show three-fold higher platelet concentrations in cats than in horses. This high concentration of platelets in cat PC compared with the results reported in horses
 can lead to longer incubation times and/or higher doses of activating substance to obtain clots in cat PC.
It is reported a TGF-β1 mean concentration of 21.48 ± 8.948 ng/mL in serum samples taken from 12 cats
. This concentration was similar to the concentrations found in the supernatants of both PC but higher than the plasma TGF-β1 concentration in this study. This discrepancy was caused by premature platelet activation. Serum differs from plasma in that the bulk of the fibrinogen has been removed by conversion into a fibrin clot together with the platelets that have either been physically bound in the fibrin matrix or activated to form aggregates or both
. This finding implies previous blood clotting and therefore platelet activation and release of the growth factors contained in the platelet alpha granules, including TGF-β1.
We were not able to find any reports on feline PDGF-BB plasma concentration. To the best of our knowledge, this study is the first time that an ELISA human kit for PDGF-BB was used to measure this protein in blood components from cats. However, it is reported that both human and cat PDGF-BB presented high peptide sequence homology
. A similar finding has also been noticed between human and equine PDGF-BB
Once the PC is prepared, platelet activation (exogenous or endogenous) may be important to maximize growth factor release. Various substances have been described for the exogenous activation of PC, including thrombin
, collagen type I
 and calcium chloride
, among others. The substances most frequently used to activate PC for clinical purposes are thrombin and calcium salts. The use of topical bovine thrombin has been reported in humans to cause the formation of antibodies against the coagulation factor V, prothrombin and thrombin
. Reports in mice show the formation of antibodies against autologous clotting factors and the induction of autoimmunity with features characteristic of systemic lupus erythematosus, including antibodies against nuclear antigens, native DNA, double-stranded DNA and cardiolipin
. For this reason, the clinical use of bovine thrombin as a platelet activator in feline medicine should be carefully studied. Reports in humans
 and horses
 investigated the use of autologous thrombin obtained by the addition of calcium gluconate to the plasma. PC activation with autologous thrombin might provide another option for clinical practice in cats, and the probability of immunological reactions would be reasonably smaller. In addition, the results of this study reported that CG has an action comparatively equipotent to BT. These reasons suggest the use of CG to induce gelation of the PC for clinical purposes in cats.