Since determination of bacterial cell number is a fundamental procedure in the field of microbiology, several methods have been developed and widely employed depending on the purpose of cell counting. SPM is one of the common methods used to estimate bacterial load [12]. The advantage of SPM is speed and convenience without additional preparation steps. On the other hand, the limitations are the inability to distinguish live bacteria from dead bacteria and a relatively narrow range of detection (10⁸–10¹⁰ bacteria/mL) [12].

A wide range of detection is not required for platelet counting in PRP preparations unlike in bacterial cell counting. However, it is more difficult to recognize platelets in PRP preparations compared to bacteria because WBCs and RBCs can more or less be included, especially when the buffy coat is included in the second spin. Lee and Tarassenko were probably inspired by the bacterial cell count and first reported the optical determination method for platelet counts [13]. However, the shortcomings of this method are that the range of RBC counts (0–3 × 10⁴/μL) is set below the RBC range (30–40 × 10⁴/μL in average) of P-PRP and that WBCs were not taken into consideration.

To solve this problem, in this study, we separated PRP preparations into two types (i.e., P-PRP and L-PRP) for evaluation and successfully validated the spectrophotometric method in P-PRP preparations. In contrast, the accuracy of this method was lower than expected in L-PRP preparations, which is reflected in the difference in the coefficient values (Fig. 4). The striking difference between P-PRP and L-PRP could be attributed to the inclusion of WBCs rather than RBCs in L-PRP as RBCs were also included in P-PRP with higher platelet counts. We speculate that WBCs were the primary factor responsible for lowering the performance and that they can disrupt light transparency more effectively than can RBCs; this is because WBCs are spherical, nucleated, and larger than disk-shaped RBCs and because the absorbance of hemoglobin contained in RBCs decreases beyond 600 nm [17] (cf., 615 nm, the peak wavelength used here). Besides counts, the size distribution of WBCs depends on individual donors. Hence, the ratios of large WBCs (e.g., neutrophils) to small WBCs (e.g., lymphocytes) widely vary across individuals, especially when they suffer from certain types of diseases, such as cancers, cardiovascular diseases, and pulmonary diseases [18,19,20,21].