A reference control composition containing a nucleated red blood cell component and the method of making are disclosed. The reference control composition includes a nucleated red blood cell component made of fixed non-nucleated blood cells and a suspension medium. The non-nucleated blood cell has a natural cell size substantially similar to a size of nucleus of said nucleated red blood cell of said blood sample. The nucleated red blood cell component can be made of equine, ovine, bovine, feline, canine, or porcine red blood cells; and it is substantially free of nucleic acid. The reference control composition can further include a white blood cell component, a red blood cell component, a platelet component, a reticulocyte component, or combinations thereof. Further disclosed are the methods of using the reference control composition for measurement of nucleated red blood cells on a blood analyzer.