The 400 microsphere per piece "rule" does not apply to all blood flow studies.

Microsphere experiments are useful in measuring regional organ perfusion as well as heterogeneity of blood flow within organs and correlation of perfusion between organ pieces at different time points. A 400 microspheres/piece "rule" is often used in planning experiments or to determine whether experiments are valid. This rule is based on the statement that 400 microspheres must lodge in a region for 95% confidence that the observed flow in the region is within 10% of the true flow. The 400 microspheres precision rule, however, only applies to measurements of perfusion to a single region or organ piece. Examples, simulations, and an animal experiment were carried out to show that good precision for measurements of heterogeneity and correlation can be obtained from many experiments with <400 microspheres/piece. Furthermore, methods were developed and tested for correcting the observed heterogeneity and correlation to remove the Poisson "noise" due to discrete microsphere measurements. The animal experiment shows adjusted values of heterogeneity and correlation that are in close agreement for measurements made with many or few microspheres/piece. Simulations demonstrate that the adjusted values are accurate for a variety of experiments with far fewer than 400 microspheres/piece. Thus the 400 microspheres rule does not apply to many experiments. A "rule of thumb" is that experiments with a total of at least 15,000 microspheres, for all pieces combined, are very likely to yield accurate estimates of heterogeneity. Experiments with a total of at least 25,000 microspheres are very likely to yield accurate estimates of correlation coefficients.