A separation assembly for enabling separation of a fluid sample into first and second phases, comprising: a collection container having a first end, a second end, and a sidewall extending therebetween, the collection container defining a longitudinal axis between the first end and the second end; and a separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) having a through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546) defined therethrough, wherein upon applied rotational force to any of the separation assembly, collection container, and separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540), the separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) transitions from a first position in which the through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546) is in an open position for fluid to pass therethrough, to a second position in which the through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546) is in a closed position for preventing fluid from being received therethrough, wherein when the separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) is in the first position, a first sealing perimeter engages the sidewall of the collection container while allowing the sample to pass through the through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546) and when the separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) is in the second position, a second sealing perimeter sealingly engages the sidewall of the collection container to prevent fluid from passing between the separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) and the sidewall and for preventing fluid from passing through the open through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546), and wherein the first sealing perimeter is different from the second sealing perimeter. a collection container having a first end, a second end, and a sidewall extending therebetween, the collection container defining a longitudinal axis between the first end and the second end; and a separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) having a through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546) defined therethrough, wherein upon applied rotational force to any of the separation assembly, collection container, and separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540), the separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) transitions from a first position in which the through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546) is in an open position for fluid to pass therethrough, to a second position in which the through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546) is in a closed position for preventing fluid from being received therethrough, wherein when the separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) is in the first position, a first sealing perimeter engages the sidewall of the collection container while allowing the sample to pass through the through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546) and when the separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) is in the second position, a second sealing perimeter sealingly engages the sidewall of the collection container to prevent fluid from passing between the separator (40, 40C, 40D, 140, 140A, 240, 240A, 340, 440, 540) and the sidewall and for preventing fluid from passing through the open through-hole (46, 46C, 46D, 146, 146A, 246, 246A, 346, 446, 546), and wherein the first sealing perimeter is different from the second sealing perimeter.