In Act V, Scene 1, Lady Macbeth has become delusional and consumed with guilt. This scene echoes Act II, Scene 2 when she was literally washing Duncan's blood off her hands. By this point (Act V, Scene 1), she is vacillating between fear and guilt. She still tries to convince herself that no one knows their crime, but simply cannot erase the guilt from mind.
What need we
Fear who knows it, when none can call our power
to account?—Yet who would have thought the old
man had so much blood in him?
Lady Macbeth is reliving the moments after Duncan’s murder. She keeps using that language of cleaning her hands, washing away the guilt and the memory. She even relives the moments after Duncan's murder and Macbeth’s immediate fear and guilt following the murder when a knock at the gate startled him (“this starting”).
What, will these hands ne’er be clean?—No more o’
that, my lord, no more o’ that: you mar all with
She is telling Macbeth (who is not there) to stop looking so guilty because it looks suspicious. She even imagines the smell of the blood in Act V, Scene 1. She can’t escape it. The language of washing hands is material and spiritual. Lady Macbeth wants to get rid of any evidence or behavioral appearance of guilt. She also is trying to erase the memory which will erase the guilt and the mental images/delusions. It is an attempt at physical, psychological and spiritual cleansing.