As described previously [7, 9], blood samples (~9.5 mL) collected without anticoagulants using vacuum plain glass tubes (A-PRF+: Jiangxi Fenglin Medical Technology Co. Ltd, Fengcheng, China) or conventional vacuum plain glass tube (Plain BD Vacutainer Tube; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) from the same donors were immediately centrifuged by an A-PRF centrifugation system (A-PRF12: Dragon Laboratory Instruments Ltd., Beijing, China) or a Medifuge centrifugation system (Silfradent S. R. L., Santa Sofia, Italy) (for the conditions of centrifugation, see Table 1). After eliminating the red blood cell (RBC) fractions, the resulting A-PRF and CGF clots were placed on dry gauze to eliminate excess amounts of serum (~10 s) and then transferred to freezing tubes for determination of growth factor contents. Frozen samples stored at −80 °C were then minced, homogenized by disposable homogenizers (BioMasher II, Nippi, Inc., Tokyo, Japan), and centrifuged at 3000 rpm for 10 min at ambient temperature. The resulting supernatants were stored at −80 °C until use.

For determination of blood cell counts, another set of A-PRF/CGF clots was prepared from blood samples obtained from the same donors.

The numbers of blood cells were determined twice in the process of PRP preparation using an automated hematology analyzer (pocH-100iV diff; Sysmex, Kobe, Japan). First, RBCs, WBCs, and platelets were counted immediately after blood collection. Second, freshly prepared PRP and PRGF samples were directly submitted for the blood cell count. The obtained values were adjusted by their relative dilution factors.

As for A-PRF and CGF preparations, RBC, WBC, and platelet counts were determined by subtracting those counts in RBC fractions, supernatant acellular serum fractions, and A-PRF/CGF exudate fractions from those counts in whole blood samples, a method which we designated “indirect subtraction method.”

The concentrations of transforming growth factor-β1 (TGF-β1), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF) in frozen PRP, PRGF, A-PRF, and CGF samples were determined using human TGF-β1, PDGF-BB, and VEGF Quantikine ELISA kits (R&D Systems, Inc., Minneapolis, MN, USA). Concentrations of interleukin-1β (IL-1β) and interleukin-6 (IL-6) were determined using the IL-1β human ELISA kit and IL-6 high sensitivity human ELISA kit (Abcam, Cambridge, MA, USA).