An Evaluation of the Accuracy of the Subtraction Method Used for Determining Platelet Counts in Advanced Platelet-Rich Fibrin and Concentrated Growth Factor Preparations

Abstract

Platelet concentrates should be quality-assured of purity and identity prior to clinical use. Unlike for the liquid form of platelet-rich plasma, platelet counts cannot be directly determined in solid fibrin clots and are instead calculated by subtracting the counts in other liquid or semi-clotted fractions from those in whole blood samples. Having long suspected the validity of this method, we herein examined the possible loss of platelets in the preparation process. Blood samples collected from healthy male donors were immediately centrifuged for advanced platelet-rich fibrin (A-PRF) and concentrated growth factors (CGF) according to recommended centrifugal protocols. Blood cells in liquid and semi-clotted fractions were directly counted. Platelets aggregated on clot surfaces were observed by scanning electron microscopy. A higher centrifugal force increased the numbers of platelets and platelet aggregates in the liquid red blood cell fraction and the semi-clotted red thrombus in the presence and absence of the anticoagulant, respectively. Nevertheless, the calculated platelet counts in A-PRF/CGF preparations were much higher than expected, rendering the currently accepted subtraction method inaccurate for determining platelet counts in fibrin clots. To ensure the quality of solid types of platelet concentrates chairside in a timely manner, a simple and accurate platelet-counting method should be developed immediately.

Keywords: concentrated growth factors; fractionation; platelet-rich fibrin; platelets; quality assurance; regenerative therapy.

Figure 1

WBC counts in the RBC fraction (a), upper plasma fraction (b), and whole blood sample (c). Peripheral blood samples were collected in the presence of the anti-coagulant and centrifuged by the individual centrifugation protocols. The upper fraction was collected above the line indicated in Table 1. The remainder of the fractionated sample was used as the RBC fraction. N = 12–15. The asterisks represent statistically significant difference (p < 0.05).

Figure 2

Platelet (PLT) counts in the RBC fraction (a), upper plasma fraction (b), and whole blood sample (c). Peripheral blood samples were collected in the presence of the anti-coagulant and centrifuged by the individual centrifugation protocols. The upper fraction was collected above the line indicated in Table 1. The rest of the fractionated sample was used as the RBC fraction. N = 12–15. The asterisks represent statistically significant difference (p < 0.05).

Figure 3

WBC (a,c) and platelet (PLT) counts (b,d) in the A-PRF (a,b) and CGF preparations (c,d). Peripheral blood samples were collected into glass tubes in the absence of the anti-coagulant and centrifuged by the individual centrifugation protocols. The freshly formed fibrin clots were withdrawn and dissected from the RBC clots. The resulting A-PRF/CGF preparations were compressed to collect the exudate fractions, whereas the RBC clots were minced and gently combined with the liquid form of RBC fraction. WBC and platelet counts were determined in the whole samples, A-PRF/CGF exudate fraction, and RBC fractions. Calculated WBC and platelet counts in the A-PRF/CGF preparations, which were calculated by the subtraction method, were substantially greater than that of the other fractions. N = 10. * The exudate fraction also included a small volume of the acellular serum fraction. ** Sum of these fractions corresponds to the upper plasma fraction shown in Table 1.

Figure 3

WBC (a,c) and platelet (PLT) counts (b,d) in the A-PRF (a,b) and CGF preparations (c,d). Peripheral blood samples were collected into glass tubes in the absence of the anti-coagulant and centrifuged by the individual centrifugation protocols. The freshly formed fibrin clots were withdrawn and dissected from the RBC clots. The resulting A-PRF/CGF preparations were compressed to collect the exudate fractions, whereas the RBC clots were minced and gently combined with the liquid form of RBC fraction. WBC and platelet counts were determined in the whole samples, A-PRF/CGF exudate fraction, and RBC fractions. Calculated WBC and platelet counts in the A-PRF/CGF preparations, which were calculated by the subtraction method, were substantially greater than that of the other fractions. N = 10. * The exudate fraction also included a small volume of the acellular serum fraction. ** Sum of these fractions corresponds to the upper plasma fraction shown in Table 1.

Figure 4

(a) Appearance of the A-PRF clot prepared using a centrifuge equipped with an angle rotor and the recommended glass tube. (b) The A-PRF preparation with a small portion of the red thrombus was prepared by scrapping off most of the red thrombus. (c) The region indicated by the yellow dot-square was subjected to examination by Scanning Electron Microscopy (SEM). In the CGF preparation, the red thrombus was usually shorter than that of the A-PRF preparation. SEM observations of aggregated platelets in the upper region of the RBC clot formed just below A-PRF preparations. Similar findings were obtained in the preparation of CGF. Bar = 10 μm.

Figure 5

WBC (a) and platelet counts (b) on the inside wall of two types of glass tubes. After the A-PRF/CGF clots prepared in the absence of the anti-coagulant were removed, the inside of the tubes were washed thoroughly. WBCs and platelets were enzymatically detached for counting. N = 8 (A-PRF+^®) or 12 (Vacutainer tube^®).

Figure 6

SEM observations of platelets on cell culture wares in the absence or the presence of RBCs. Washed platelets suspended in Hepes–Tyrode solution were placed on plastic dishes (a), hydrophobic lids of plastic dishes (b) or glass coverslips (c) and incubated for 10 min at 37 °C. As above, RBC-contaminated platelet suspensions were plated on the coverslip and incubated (d). Data is representative of three independent experiments. Bar = 10 μm.

Figure 7

Scheme of migration of major blood components during centrifugation.