A computer simulation study to produce ultrasonic backscatter coefficients (BSCs) from red blood cell (RBC) clusters is discussed. The simulation algorithm is suitable for generating non-overlapping, isotropic, and fairly identical RBC clusters. RBCs were stacked following the hexagonal close packing (HCP) structure to form a compact spherical aggregate. Such an aggregate was repeated and placed randomly under non-overlapping condition in the three-dimensional space to mimic an aggregated blood sample. BSCs were computed between 750 KHz and 200 MHz for samples of various cluster sizes at different hematocrits. Magnitudes of BSCs increased with mean aggregate sizes at low frequencies (<20 MHz). The accuracy of the structure-factor-size-estimator (SFSE) method in determining mean aggregate size and packing factor was also examined. A good correlation (R2 ≥ 0.94) between the mean size of aggregates predicted by the SFSE and true size was found for each hematocrit. This study shows that for spherical aggregates there exists a region for each hematocrit where SFSE works most accurately. Typically, error of SFSE in estimating mean cluster size was <20% for dimensions between 14 and 17 μm at 40% hematocrit. This study suggests that the theoretical framework of SFSE is valid under the assumption of isotropic aggregates
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