Methods for segregating particles using an apparatus with a size-discriminating separation element having an elongate leading edge
The disclosure relates to an apparatus for segregating particles on the basis of their ability to flow through a stepped passageway. At least some of the particles are unable to pass through a narrower passageway bounded by a segregating step, resulting in segregation of the particles. The breadth of the leading edge of at least one step of the apparatus is significantly greater than the overall width of the passageway in which the step occurs, permitting high and rapid sample throughput. The apparatus and methods described herein can be used to segregate particles of a wide variety of types. By way of example, they can be used to segregate circulating tumor cells from a human blood sample.
1. A method of segregating circulating tumor cells (CTCs) from blood cells in a whole blood sample, wherein said method employs a device comprising:
a body and
a cover that define a void therebetween, the void containing
a separation element that segregates an inlet region and an outlet region of the void, the separation element defining, together with a surface of the void,
a channel that fluidly connects the inlet and outlet regions by way of a separating portion into which the separation element projects, the channel having
an overall width at the separating portion and
a height defined by the distance between the separation element and the surface of the void,
at least one of the body, the cover or the separation element bearing a segregating step disposed within and having a leading edge extending substantially completely across the separating portion of the channel, whereby the channel is divided into an upstream portion on the inlet side of the leading edge and a substantially lamellar downstream portion on the outlet side of the leading edge, the upstream portion of the channel being also lamellar in a region between the inlet region and the separation element; the height of the upstream portion being sufficient to facilitate passage therethrough of both said CTCs and said blood cells,
the height of the downstream portion being sufficiently large to facilitate passage therethrough of said blood cells and sufficiently small to inhibit passage therethrough of said CTCs, and
the leading edge having a length at least 20 times greater than the overall width of the channel at the separating portion such that the leading edge has an undulating shape;
and said method comprises
urging said whole blood sample through said channel of said device from the inlet region whereby CTCs will be segregated from said blood cells due to their characteristics and inability to traverse said segregating step along the separating portion of the channel.
2. The method as claimed in claim 1 which further comprises recovering said segregated CTCs that are unable to pass to the outlet region.
3. The method as claimed in claim 2 which further comprises backflushing fluid from the outlet region in the direction of the inlet region to recover said segregated CTCs.
4. The method as claimed in claim 1 wherein said CTCs are derived from a solid tumor.
5. The method as claimed in claim 4 wherein said CTCs are derived from an ovarian, prostate or breast cancer tumor.
6. The method as claimed in claim 1 wherein said device has a size approximately equal to a microscope slide.
7. The method as claimed in claim 1 wherein said leading edge has a serpentine shape.
8. The method as claimed in claim 1 wherein the separation element of said device includes a plurality of segregating steps disposed serially within the separating portion, each segregating step:
(a) having a leading edge that extends substantially across the separating portion and having a length substantially greater than the overall width of the channel at the separating portion; and
(b) dividing the channel into an upstream portion and a substantially lamellar downstream portion relative to the leading edge of the segregating step, the height of the channel at the downstream portion immediately following the segregating step being smaller than the height of the channel at the upstream portion immediately preceding the segregating step.
9. A method of diagnosing occurrence of a tumor in a vertebrate subject, the method comprising the steps of:
(i) segregating CTCs from a blood sample obtained from the subject using a method according to claim 1 ; and
(ii) determining the occurrence of thus segregated CTCs, wherein said occurrence of segregated CTCs is indicative of occurrence of a tumor.
10. A method of assessing efficacy of a tumor treatment in a subject afflicted with a tumor, the method comprising the steps of:
segregating CTCs from blood samples obtained from the subject before and after the treatment using a method according to claim 1 and
(ii) comparing the concentration of CTCs in the samples from the CTCs thus segregated, whereby decrease of the concentration of CTCs after treatment is indicative of its effectiveness.