Process, device and reagent for cell separation

• US 20020028431A1
• Filed: 02/22/2001
• Published: 03/07/2002
• Est. Priority Date: 08/25/1998
• Status: Active Grant

• 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: Julien, Jean-Claude Bisconte de Saint
• Granted Patent: US 7,316,897 B2
• Time in Patent Office: Days
• 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 the isolation of pathogenic cells in a small concentration in a biological fluid specimen, comprising:

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.

2. The cell separation process according to claim 1, wherein said differential migration is caused by filtration of the biological fluid specimen and performed by a filter with a pore size between 5 and 10 μ

m.

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

m.

4. The cell separation process according to any one of claims 2-3, further comprising a specimen-preparation step performed prior to filtration which comprises adding to the blood specimen a reagent for lysis of red cells in the fluid specimen and fixation of nucleated cells.

5. The cell separation process according to any one of claims 2-3, further comprising a specimen-preparation step performed prior to filtration which comprises adding to the fluid specimen a reagent comprising EDTA, bovine serum albumin, saponin, formaldehyde and PBS.

6. The cell separation process according to any one of claims 2-3, further comprising a specimen-preparation step performed prior to filtration which comprises adding to one fluid specimen unit nine units of reagent for lysis of red cells and fixation of nucleated cells, and incubating the resulting preparation for approximately 15 minutes.

7. The cell separation process according to any one of claims 2-3, wherein filtration is performed under a partial vacuum of approximately 50,000 Pa.

10. A reagent for cell separation according to the process in accordance with at least one of claims 1 or 8, comprising a detergent which degrades the lipid membrane of red cells in the specimen and a fixative that hardens the membrane of nucleated cells.

11. The reagent according to claim 10, comprising at least one of EDTA, BSA, saponin, formaldehyde and PBS.

12. The reagent according to claim 11, comprising 372 mg of EDTA, 1 g of BSA, 1.75 g of saponin, 10 ml of 37% formaldehyde solution and a sufficient amount of PBS to yield 1 liter of reagent.

14. The device according to claim 13, wherein the filtration membrane has a pore size of approximately 8 μ

m.

15. The device according to claim 13 or 14, further comprising means for creating a flow essentially tangential to a surface of the filter.

16. Application of the separation process according to claims 1 or 8 for the detection of micrometastases in a blood specimen.

17. Application of the separation process according to claims 1 or 8 for the detection of cells infected by a virus in a biological fluid.

18. A device for counting target cells isolated by the process according to claims 1 or 8, comprising a filter with a pore size of approximately 8 microns and a broad-field photon counting system.

19. A device for counting target cells isolated by the process according to claims 1 or 8, comprising a filter with a pore size of approximately 8 microns and a means for image analysis of the filtrate.

20. A device enabling the selection and the individual collection of cells for the purpose of subsequent characterization and according to the process in claims 1 or 8, comprising a filter with a pore size adapted to the size of the target cells.

8. A process for the detection of cells capable of being present in a very low concentration in a biological fluid specimen comprising:

reacting a reagent specific to particles to be detected in the specimen or, specific to particles normally present in the biological specimen and capable of masking the presence of targeted cells to modify physical properties of the cells that reacted;

performing an enrichment of a part of the specimen by a physical selection process ensuring differential migration of the particles that reacted with the reagent versus the particles that did not react;

performing a visualization of the possible presence of the targeted particles in the part of the specimen enriched in targeted particles.

9. The process according to claim 8, wherein a reagent is reacted which modifies the relative deformability of the targeted cells in relation to the deformability of the cells capable of masking the targeted cells.

13. A device for the separation of pathogenic cells in a blood specimen, comprising a filtration membrane with a pore size between 5 and 10 μ

m.