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The following notes apply to cytosphere counting (Coulter, Hialeah, FL). A blocking reagent with CD14 monoclonal antibody-labeled beads is added to the CD4 1 tube to achieve this objective. CD14 beads are added to whole blood and incubated under constant shaking. The tubes are shaken and not vortexed, as vortexing creates forces which cause the beads to disassociate from their respective cells. After the monocytes have been tagged, CD4 beads are added to the mixture. The process is repeated with the CD8 1 tube except for the monocyte-blocking step.