Evaluation of methods for sizing and counting of ultrasound contrast agents.

A precise, accurate and well documented method for the sizing and counting of microbubbles is essential for all aspects of quantitative microbubble-enhanced ultrasound imaging. The efficacy of (a) electro-impedance volumetric zone sensing (ES) also called a Coulter counter/multisizer; (b) optical microscopy (OM); and (c) laser diffraction (LD), for the sizing and counting of microbubbles was assessed. Microspheres with certified mean diameter and number concentration were used to assess sizing and counting reproducibility (precision) and reliability (accuracy) of ES, OM and LD. SonoVue™ was repeatedly (n = 3) sized and counted to validate ES, OM and LD sizing and counting efficacy. Statistical analyses of intra-method variability for the SonoVue™ mean diameter showed that the best microbubble sizing reproducibility was obtained using OM with a mean diameter sizing variability of 1.1%, compared with a variability of 4.3% for ES and 7.1% for LD. The best microbubble counting reproducibility was obtained using ES with a number concentration variability of 8.3%, compared with a variability of 22.4% for OM and 32% for LD. This study showed that no method is fully suited to both sizing and counting of microbubbles.