**7. The methods of obtaining platelet-rich plasma**

The two main methods of obtaining platelets are by autologous donation and by the technique of platelet apheresis. The advantage of the apheresis technique is that the platelet concentration is higher in the final concentrate and should contain at least 5.5 × 10 10 platelets, while its disadvantage is the cost that is extremely superior to the autologous total donation technique [48].

In the apheresis technique for platelet collection, Rezende et al. used the Haemonetics MCS + 9000 automatic cell separator and the 995-E apheresis-specific kit (Haemonetics Corp.). In that system, by means of a venipuncture, the blood of the patient himself is drained to a separation device. An optical refraction analyzer separates the platelet layer, and the remaining blood is completely returned to the patient, determining the end of a cycle. Sodium citrate can be used as an anticoagulant in the ratio of 1 to every 9 ml of whole blood processed. In 2 cycles, 72 ml of platelet concentrate was collected; the hematimetric indices of the patient and also the platelet concentrate indices are evaluated before and after the procedure (Coulter AcT Diff) [81].

Platelet growth factors were generally obtained in a ranked room; hence, the handling of platelet concentrate was performed within a Category II-Type A biological safety cabinet. Then, 2800 microliters of 10% calcium chloride was added, and the final product was maintained at +37°C for approximately 30 min. Subsequently, the unit was subjected to centrifugation (900G), and the supernatant serum, which contained the platelet growth factors, was transferred to four 50 ml falcon-type tubes (Becton-Dickinson) and maintained at −80°C (Revco). Research of bacteria, aerobic and anaerobic, and fungal agents was performed in a systematic way.

The release of serum with platelet growth factors in the study by Rezende et al. was done as follows: weekly, thawed Falcon tube containing approximately 10 ml of autologous serum with platelet growth factors that was transferred to flasks, and the patient is advised to keep the biological medicine at a temperature below −10°C (freezer) and to unfreeze it naturally immediately before each use [81].

Alio et al. used the autologous donation technique to obtain platelets, when patients were submitted to venipuncture and 80–100 ml of blood were collected in sterile 10 ml tubes containing 1 ml of sodium citrate to avoid coagulation. These tubes were left at room temperature for 10 min, and only the supernatant (upper tube fraction) was collected as the final product. The platelet concentrate was then prepared under sterile conditions in a laminar flow room. Two to three milliliters of this concentrate was then placed in sterile eyedrops. The eyedrops were kept at −20°C, and only when the patient was going to use an eyedrop bottle, thaw it and then keep it at +4°C, discarding that bottle at the end of a week, when a new one is thawed. The patients were advised to use these eyedrops four times a day for 1 month [88].

Both methods require the patient to perform an autologous blood donation. Autologous blood does not transmit disease; there is no occurrence of hemolytic (alloimmunization), allergic, immunological (immunomodulation), or acute lung injury by transfusion and common complications in heterologous donations [91].

The contraindications for autologous donations are anemia or other types of pathological hemodilution; conditions that lead to oxygen saturation and hemoglobin saturation (less than 11 mg/dl); hepatopathies; nephropathies; coagulopathies; hemoglobinopathies; decompensated heart diseases; and presence of infectious diseases such as Chagas, syphilis, HIV, HTLV, hepatitis B and C, and others transmissible by blood considered as "relative" contraindications, since the patient will receive the same, but there may be contamination of the health team in the handling of that blood. In HIV, there may be reactivation of the virus when reinfused [91, 92].

Complications of autologous donation are those inherent to a donor, such as hypotension, anemia, angina, and contamination of the material of the blood bags [91].
