Reference control compositions and the method of use are disclosed for measurement of nucleated red blood cells, which includes one set of synthetic spherical particles having a mean particle diameter ranging from 6.2 m to 6.8 m and a refractive index from 1.58 to 1.62 monodispersed in an aqueous suspension medium. The synthetic spherical particles have optical properties simulating optical properties of nucleated red blood cells as measured by optical measurements. The reference control composition can further include a second set of the synthetic spherical particles having optical properties simulating optical properties of white blood cells. Further disclosed is a reference control system which includes a series of reference control compositions, each having an amount of one type of synthetic spherical particles which have optical properties simulating the optical properties of nucleated red blood cells having a specific cell maturity.