Injectable autologous platelet-rich plasma for regenerative medicine in donkeys

A step-by-step procedure for autologous platelet-rich plasma production was developed for topical percutaneous injection in donkeys. This protocol was used in the following publication:

The use of non-transfusional hemocomponents for tissue healing has gained increasing popularity for the treatment of musculoskeletal lesions in human and veterinary medicine [1].Several nontransfusional hemocomponents are available for intralesional injection, including platelet-rich plasma (PRP), plasma rich in growth factors, platelet rich fibrin, platelet lysate, autologous conditioned serum, autologous blood preparations and autologous protein concentrate [2][3][4].PRP is a good adjunctive therapy for the treatment of orthopedic and soft tissue conditions [5][6][7][8][9][10][11]. Nonunions, bone defects, tendinosis and cartilage defects are among musculoskeletal conditions lacking effective treatment modalities, and regenerative medicine may play an important role.Platelet rich plasma contains a variety of growth factors released from platelets, which increase vascular growth and have mitogenic effects on mesenchymal stem cells [12][13][14][15][16]. Clinical research on donkeys needs to be in continual development, since donkeys have different reactions in many conditions when compared to horses [17].To our knowledge, PRP production and application is not commonly performed in donkeys, despite the high therapeutic potential of PRP application in this species.This protocol was used in the following publication: Collect autologous whole blood (50 ml) from the jugular vein into a 60mL syringe.Add acid citrate dextrose solution (ACD-A) at a ratio of 1:9 achieving anticoagulation.ACD-A solution contains sodium citrate bihydrate 22.0 g/L, citric acid monohydrate 8.0 g/L, glucose monohydrate 24.5 g/L in sterile water for injection.Additionally, collect 10.5 ml whole blood in sodium citrate tubes (3.8%) to extract thrombin.
Complete blood count 3. Use a small aliquot of whole blood for complete blood cell count.

4.
For density separation of blood components, transfer the 50 ml specimen to a Falcon tube and spin at 350 units of gravitational force (x g) for 20 min.
First separation of blood components

5.
Separate plasma and buffy coat layer and transfer in a Falcon tube under aseptic conditions in a laminar flow cabinet.
Second centrifugation

6.
Spin the plasma and the buffy coat again at 900 x g for 15 min to separate the platelet pellet, in the bottom layer, from the platelet poor plasma (PPP) in the supernatant layer.
Second separation of blood components 7. Discard part of the PPP, leaving in the tube 10mL volume.
Re-suspension of the solution 8. Resuspend the platelet pellet in the PPP to obtain 10 ml of PRP.
PRP cell count

9.
Perform cellular count from PRP automatically.Compare the mean platelet concentration in the PRP and in the whole blood.
To obtain the thrombin, mix the autologous plasma fraction and 10% calcium gluconate (446 mEq/l of calcium), at a ratio of 5:1, and incubate at 37°C for 30 min, in an air-jacketed CO 2 incubator.Squash the clot obtained and collect the final supernatant, the thrombin-rich solution.

11.
Activate the PRP by mixing the PRP and the thrombin-rich solution (volumetric ratio 8:1) in a Falcon tube and gently rotate the tube.
Recommendations for laboratory conditions during the production phases

12.
Perform these laboratory procedures under aseptic conditions in a laminar flow cabinet following Good Laboratory Practice.
Sterility assay of the PRP product

13.
Evaluate aerobic, anaerobic and fungal contaminations by bacteriological and mycological exams of the PRP product.
Topical application of the PRP

14.
Inject the PRP percutaneously in the target site, after application of routine aseptic skin preparation procedure.When necessary, the use of a guidance technique (e.g.diagnostic imaging) is recommended to accurately reach the appropriate injection site or the site of injury.