3.2. Sample preparation

90 human blood were used to validate the feasibility of this system. These discarded and anonymized samples were collected under protocol number 2019KY-167, approved by the Ethical Review Board of the First Affiliated Hospital of the University of Science and Technology of China. The clinical samples were run on an automated, flow-based hematology analyzer (BC6800, mindray, China) in the First Affiliated Hosptial of University of Science and Technology of China, generating a CBC report whose values for MCV, MCHC, and RDW are regarded as ground truth. RDW is typically parameterized as the standard deviation (RDW-SD), expressed in femtoliters, or the coefficient of variation (RDW-CV), expressed as a percent. In this case, we utilize the RDW -CV from the clinical report. Following this, samples were stored in an incubator and transported to the laboratory for further testing using our method. During testing, approximately 5 μL of blood was taken from the tube with an Eppendorf pipette and diluted 200-fold in a premixed solution of phosphate buffered saline (PBS) containing Cocamidopropyl betaine (CAPB, 80 μg/mL final concentration) and gently shaken for about 30 seconds. CAPB is a zwitterionic surfactant that intercalates within the red cell membrane, lowering the surface tension and causing the cell to change its shape from a biconcave disk to a sphere, while preserving its volume. This method was pioneered by Kim and Ornstein [39], and expanded to zwitterionic surfactants by Fan et al. [45]. This method has been validated and is currently standard practice on all hematology analyzers that utilize light scattering for cell volume determination [46,47]. We have further validated the sphering in our own laboratory as described previously [27]. To ensure proper sphering, samples were allowed to stand about 3 min. Later, a 10 μL aliquot of each diluted sample was placed in a sample chamber ({"type":"entrez-nucleotide","attrs":{"text":"C10283","term_id":"56146369","term_text":"C10283"}}C10283, Thermo Fisher, USA, chamber height 100 ± 2 μm) and measured immediately.