Process, device and reagent for cell separation

• US 7,316,897 B2
• Filed: 02/22/2001
• Issued: 01/08/2008
• Est. Priority Date: 08/25/1998
• Status: Expired due to Term

• Current Assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm) (Allianz Global Investors GmbH), Hopitaux De Paris L'Assistance Publique, L'Universite RENE Descartes Paris 5
• Original Assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm) (Allianz Global Investors GmbH), Hopitaux De Paris L'Assistance Publique, L'Universite RENE Descartes Paris 5
• Inventors: Paterlini, Patricia, Bisconte de Saint Julien, Jean-Claude
• Primary Examiner(s): Shibuya; Mark L.
• Application Number:
  US09/790,673
• Publication Number:
  US 20020028431A1
• Time in Patent Office: 2,511 Days
• Field of Search: 436/17, 436/18, 436/64, 436/177, 435/2, 435/6, 435/173.7, 435/5, 435/7.23, 435/366, 435/371, 435/372
• US Class Current: 435/2
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/5761 Hepatitis B
• CPC Class Codes: G01N 33/5767 non-A, non-B hepatitis

Abstract

A cell separation process for the isolation of pathogenic cells in a small concentration in a biological fluid specimen, including treating the biological specimen to modify in a differential manner selected rare cells and other cells and causing differential migration of cells that reacted during treatment versus cells that did not react during treatment.

Claims

1. A cell separation process for isolating at least one of target cells selected from the group consisting of epithelial cells, endothelial cells, micrometastases and cells infected by a virus in small concentration in a blood specimen, comprising:

chemically treating the blood specimen with a reagent to modify relative deformability of the target cells;

adding to the blood specimen a reagent for lysis of red cells and fixation of nucleated cells; and

separating the target cells by filtration with a filter with a pore size between 5 and 10 μ

m of the blood specimen based on the lack of deformability of the rigidified target cells versus cells that maintained deformability during chemical treatment.

2. The cell separation process according to claim 1, wherein filtration of the blood specimen is performed with a filter with a pore size of approximately 8 μ

m.

6. The cell separation process according to any of claims 1-4, wherein the target cells are cells infected by a virus.

7. The cell separation process according to any of claims 1-4, wherein filtration of the blood specimen is performed on a polycarbonate film.

3. A cell separation process for isolating cells selected from the group consisting of epithelial cells, endothelial cells, micrometastases and cells infected by a virus in small concentration in a blood specimen, comprising:

chemically treating the blood specimen with a reagent to modify relative deformability of the target cells; and

separating the target cells by filtration with a filter with a pore size between 5 and 10 μ

m of the blood specimen based on the lack of deformability of the rigidified target cells versus cells that maintained deformability during chemical treatment,

and wherein the reagent comprises EDTA, bovine serum albumin, saponin, formaldehyde and PBS.

4. A cell separation process for isolating at least one of target cells selected from the group consisting of epithelial cells, endothelial cells, micrometastases and cells infected by a virus in small concentration in a blood specimen, comprising:

chemically treating one unit of blood specimen with a reagent to modify relative deformability of the target cells;

adding to one blood specimen nine units of reagent for lysis of red cells and fixation of nucleated cells, and incubating the resulting preparation for approximately 15 minutes; and

separating the target cells by filtration with a filter with a pore size between 5 and 10 μ

m of the blood specimen based on the lack of deformability of the rigidified target cells versus cells that maintained deformability during chemical treatment

5. The cell separation process according to any of claims 4, wherein the target cells are micrometastatic cells.

8. A cell separation process for isolating at least one of target cells selected from the group consisting of epithelial cells, endothelial cells, micrometastases and cells infected by a virus in small concentration in a blood specimen, comprising:

chemically treating the blood specimen with a reagent to modify relative deformability of the target cells; and

separating the target cells by filtration of the blood specimen based on the lack of deformability of the target cells versus cells that maintained deformability during chemical treatment, wherein filtration of the blood specimen is performed with a filter subjected to a partial vacuum on the order of 50,000 Pa.

9. The cell separation process of claim 8, wherein the target cells are cytokeratin-rich cells.

10. A cell separation process for isolating at least one of target cells selected from the group consisting of epithelial cells, endothelial cells, micrometastases and cells infected by a virus in small concentration in a biological fluid specimen, comprising:

chemically treating the biological fluid specimen with a reagent to modify relative deformability of the target cells; and

separating the target cells by filtration with a filter with a pore size between 5 and 10 μ

m of the biological fluid specimen based on the lack of deformability of the rigidified target cells versus cell'"'"'s that maintained deformability during chemical treatment,

wherein the target cells are cytokeratin-rich cells or virus-infected cells, and the biological fluid sample is other than a blood sample, and wherein the reagent comprises EDTA, bovine serum albumin, saponin, formaldehyde and PBS.

11. The cell separation process of claim 10, wherein the biological fluid sample comprises cerebrospinal fluid, lymph fluid or articular fluid.

12. The cell separation process according to claim 10, wherein said separation is caused by filtration of the biological fluid specimen with a filter with a pore size between 5 and 10 μ

m.

13. The cell separation process according to claim 10, wherein filtration of the biological fluid specimen is performed with a filter with a pore size of approximately 8 μ

m.

14. The cell separation process of claim 10, wherein filtration of the biological fluid specimen is performed on a polycarbonate film.

15. A cell separation process for isolating at least one of target cells selected from the group consisting of epithelial cells, endothelial cells, micrometastases and cells infected by a virus in small concentration in a biological fluid specimen, comprising:

chemically treating the biological fluid specimen with a reagent to modify relative deformability of the target cells; and

separating the target cells by filtration with a filter with a pore size between 5 and 10 μ

m of the biological fluid specimen based on the lack of deformability of the rigidified target cells versus cells that maintained deformability during chemical treatment,

wherein the target cells are cytokeratin-rich cells or virus-infected cells, and the biological fluid sample is other than a blood sample, and wherein filtration of the biological fluid specimen is performed with a filter subjected to a partial vacuum on the order of 50,000 Pa.

16. The cell separation process of claim 15, wherein the biological fluid sample comprises cerebrospinal fluid, lymph fluid or articular fluid.

17. The cell separation process according to claim 15, wherein said separation is caused by filtration of the biological fluid specimen with a filter with a pore size between 5 and 10 μ

m.

18. The cell separation process according to claim 15, wherein filtration of the biological fluid specimen is performed with a filter with a pore size of approximately 8 μ

m.

19. The cell separation process of claim 15, wherein filtration of the biological fluid specimen is performed on a polycarbonate film.