Blood samples were collected from 11 non-smoking healthy male volunteers aged 33 to 69 years. The study design and consent forms for all the procedures were approved by the ethics committee for human participants at the Niigata University School of Medicine (Niigata, Japan) in accordance with the Helsinki Declaration of 1964 as revised in 2013.

Peripheral blood (~ 9 mL) was collected into plastic vacuum plain blood collection tubes (Neotube; NIPRO, Osaka, Japan) containing 1 mL of the A-formulation of acid-citrate-dextrose (ACD-A; Terumo, Tokyo, Japan). The whole-blood samples were stored using a rotating agitator at ambient temperature and were used within 36 h. The whole-blood samples were centrifuged at 533×g for 10 min (first low-speed spin). For P-PRP preparation, the upper plasma fraction, which was approximately 2 mm beyond the interface between the plasma and RBC fractions, was transferred into 2-mL sample tubes for the second high-speed spin (2656×g, 5 min). For L-PRP preparation, the upper plasma fraction was transferred along with a buffy coat and the surface of the RBC fraction for the second spin. Prior to the second spin, 0.5 μg/mL prostaglandin E₁ (PGE₁) (Wako Pure Chemicals, Osaka, Japan) was added to each sample to prevent platelet aggregation. After centrifugation, 50–70% of the supernatant (PPP) was removed, and platelets (and other blood cells, if any) were resuspended in the remaining PPP fraction.

The numbers of platelets and other blood cells in the whole-blood samples and PRP preparations were determined using an AHA (pocH 100iV, Sysmex, Kobe, Japan).

P-PRP and L-PRP preparations were serially diluted with the corresponding amount of PPP. The series of P-PRP and L-PRP dilutions were first subjected to measurement using the AHA and subsequently subjected to measurement with a compact scanning probe microscope (SPM; PiCOSCOPE, Ushio Inc., Tokyo, Japan) (Fig. 1). The SPM can be operated by remote control through a specific application installed on smart devices, including the iPad Air (Apple, Cupertino, CA, USA). PRP samples were transferred into 0.2 mL highly transparent PCR tubes (Nippon Genetics Co., Ltd., Tokyo, Japan) and were measured at 615 nm (range of wavelength 570–660 nm).