Just like in the main game, you will encounter several Little Sisters throughout Minerva's Den. There are a total of six Little Sisters, three within Minerva's Den, and three within Operations. For the achievement, you must deal with all six of the Little Sisters found through the DLC. It doesn't matter whether you Save or Harvest all or some of them, so long as you deal with all of them.
If you still need to rescue a Sister, but she doesn’t appear anywhere on the map, walk around the area until you see a Lancer. Follow him to a ventilation hole where he will call a Little Sister, were you will then be able to rescue/harvest her.
Note: If you leave Minerva's Den or Operations and you haven't rescued/harvested the Sisters three Little Sisters here, you WON'T be able to come back!