Imaging of immunomagnetically enriched rare cells
View Patent ↗A method for removing excess unbound ferrofluid and imaging immunomagnetically enriched circulating tumor cells is provided. A vessels having a preformed grooves in the viewing surface is optimally designed for cell alignment and imaging. After separating the unbound particles by centrifugation, an externally-applied force is applied to transport magnetically responsive particle-CTC complex toward the transparent collection wall. The grooved inner surface of the viewing face of the chamber provide uniform distribution of the particles for easy imaging. The invention is also useful in conducting quantitative analysis and sample preparation in conjunction with automated cell enumeration techniques as in quantitative analysis of CTC in disease.
1. An improved method for optically analyzing rare cells suspended in a fluid medium, which method comprises:
a. obtaining a blood sample from a patient suspected of having cancer;
b. mixing said blood sample with ferrofluid particles linked to an antibody specific for the epithelial cell adhesion molecule_antigen on the surface of epithelial cells;
c. removing unbound ferrofluid from bound ferrofluid; and
d. viewing magnetically responsive constituents in the blood sample using Time Delay Integration in a viewing chamber wherein said constituents are uniformly-distributed within a preformed alignment structure containing grooves having an image viewing surface and an alignment surface on the inner surface of the optically-transparent face of said chamber.
2. The method of claim 1 wherein said alignment structures are foxhole-shaped grooves.
3. The method of claim 1 wherein said image viewing surface is 80 um.
4. The method of claim 1 wherein said removing unbound ferrofluid comprises:
a. spinning said blood sample with ferrofluid particles in a conical tube at 900 rpm;
b. aspirating the sample from the bottom of said conical tube; and
c. repeat steps (a) and (b) 5 times.
5. The method of claim 1 wherein said viewing further includes confirming the bound epithelial cell as a circulating tumor cells.
6. The method of claim 5 wherein said confirming comprises:
a. positive first labeling of the nucleus with 4′,6-diamidino-2-phenylindole;
b. positive second labeling of the cytoskeleton with Cytokeratine phycoerythrin; and
c. positive third labeling of leukocytes with CD45 allophycocyanin.
7. The method of claim 6 further including bright field examining of captured circulating tumor cells.