Developed by Dr. Alberto Orfao and colleagues in Spain, AccuCheck Counting Beads are an efficient single platform method for absolute cell counts that combines the advantages of direct flow cytometric immunophenotyping with the use of two different fluorescent beads (A and B beads). These two fluorospheres are used as a double internal standard for blood volume calculation. A known volume of AccuCheck Counting Beads is added to the same known volume of stained blood in a lyse-no-wash technique. The beads are counted along with cells. Since the concentration of bead unknown, the number of cells per microliter (the absolute count) is obtained by relating the number of cells counted to the total number of fluorescent bead events. The cell number is then multiplied by the number of total fluorospheres per unit of volume. As the Pipette Check Counting Bead system contains two different fluorospheres in a known proportion, we can first assure the accuracy of the assay by verifying the proportion of both types of beads. The final absolute count is determined as describes in the following process:

Final Absolute Count = (number of cells counted / total number of beads counted (A+B)) x number of beads per µl (known concentration) 

Beads A and B have been designed such that their unique characteristics allow the determination of whether they have been homogenously sampled. Bead A is a 6.4µM sphere that in flow cytometry presents a low forward scatter (FSC) signal, a lower side scatter (SSC) signal, and emits broadly when excited with a 488 nm argon laser; however, bead A does not fluoresce when excited with either a 633 nm HeNe or 635 nrn red diode laser. Bead B is a 6.36 µm sphere that presents a low FSC signal, a slightly higher SSC signal and a higher fluorescent signal when excited with a 488 nm argon 
laser. Unlike bead A, bead B fluoresces when excited by a longer wavelength red laser.