Analysis of Circulating Tumor Cells, Fragments, and Debris

• US 20110104718A1
• Filed: 11/02/2010
• Published: 05/05/2011
• Est. Priority Date: 08/23/2001
• Status: Active Grant

• Current Assignee: Menarini Silicon Biosystems S.P.A
• Original Assignee: Veridex LLC (Johnson & Johnson)
• Inventors: O'Hara, Shawn Mark, Rao, Galla Chandra, Repollet, Madeline, Rutner, Herman, Larson, Christopher, Gross, Steven, Terstappen, Leon W.M.M.
• Granted Patent: US 8,329,422 B2
• Time in Patent Office: Days
• US Class Current: 435/7.23
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: Y10S 436/813 Cancer
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/25125 Digestion or removing interfering materials
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

The methods and reagents described in this invention are used to analyze circulating tumor cells, clusters, fragments, and debris. Analysis is performed with a number of platforms, including flow cytometry and the CellSpotter® fluorescent microscopy imaging system. Analyzing damaged cells has shown to be important. However, there are two sources of damage: in vivo and in vitro. Damage in vivo occurs by apoptosis, necrosis, or immune response. Damage in vitro occurs during sample acquisition, handling, transport, processing, or analysis. It is therefore desirable to confine, reduce, eliminate, or at least qualify in vitro damage to prevent it from interfering in analysis. Described herein are methods to diagnose, monitor, and screen disease based on circulating rare cells, including malignancy as determined by CTC, clusters, fragments, and debris. Also provided are kits for assaying biological specimens using these methods.

Claims

1. A method for diagnosing disease in a test subject comprising:

a. obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing intact rare cells and further comprising;

i. cell fragments derived from rare cells, or

ii. cellular debris derived from rare cells;

b. preparing a magnetically-labeled sample wherein said biological sample is mixed with magnetic particles coupled to a first biospecific ligand which reacts specifically with said intact rare cells, and said cell fragments or said cellular debris, to the substantial exclusion of other specimen components;

c. contacting said magnetically-labeled sample with at least one additional biospecific ligand which specifically labels said intact rare cells, and said cell fragments or said cellular debris, to the substantial exclusion of other specimen components;

d. analyzing said labeled rare cells, and said labeled cell fragments or said labeled cellular debris, the presence of said labeled rare cells, said labeled cell fragments, and said labeled cellular debris indicating the presence of disease.

2. The method of claim 1, wherein said biological specimen is blood.

3. The method of claim 2, wherein after said biological specimen obtained, it is contacted with an agent capable of stabilizing said biological specimen.

4. The method of claim 1, wherein said magnetic particles are colloidal.

5. The method of claim 1, wherein after the step of preparing said magnetically-labeled sample, said sample is subjected to a high gradient magnetic field to produce a separated magnetically-labeled fraction which is enriched for said intact rare cells, and said cell fragments or said cellular debris.

6. The method of claim 1, wherein said analysis is selected from the group consisting of:

multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, and automated cell analysis.

7. The method of claim 1, wherein said analysis is based on at least one of the group consisting of:

morphologic analysis and epitopic analysis.

8. A method for diagnosing disease in a test subject comprising:

a. obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing intact rare cells and clusters of rare cells;

b. preparing a magnetically-labeled sample wherein said biological sample is mixed with magnetic particles coupled to a first biospecific ligand which reacts specifically with said intact rare cells and said clusters of rare cells, to the substantial exclusion of other specimen components;

c. contacting said magnetically-labeled sample with at least one additional biospecific ligand which specifically labels said intact rare cells and said clusters of rare cells, to the substantial exclusion of other specimen components;

d. analyzing said labeled rare cells and said labeled clusters of rare cells, the presence of said labeled rare cells and said labeled clusters of rare cells indicating the presence of disease.

9. The method of claim 8, wherein said biological specimen is blood.

10. The method of claim 9, wherein after said biological specimen obtained, it is contacted with an agent capable of stabilizing said biological specimen.

11. The method of claim 8, wherein said magnetic particles are colloidal.

12. The method of claim 8, wherein after the step of preparing said magnetically-labeled sample, said sample is subjected to a high gradient magnetic field to produce a separated magnetically-labeled fraction which is enriched for said intact rare cells and said clusters of rare cells.

13. The method of claim 8, wherein said analysis is selected from the group consisting of:

multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, and automated cell analysis.

14. A method for diagnosing malignancy in a test subject comprising:

a. obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing intact malignant cells and further comprising;

i. cell fragments derived from malignant cells, or

ii. cellular debris derived from malignant cells;

b. preparing a magnetically-labeled sample wherein said biological sample is mixed with magnetic particles coupled to a first biospecific ligand which reacts specifically with said intact malignant cells, and said cell fragments or said cellular debris, to the substantial exclusion of other specimen components;

c. contacting said magnetically-labeled sample with at least one additional biospecific ligand which specifically labels said intact malignant cells, and said cell fragments or said cellular debris, to the substantial exclusion of other specimen components;

d. analyzing said labeled malignant cells, and said labeled cell fragments or said labeled cellular debris, the presence of said labeled malignant cells, said labeled cell fragments, and said labeled cellular debris indicating the presence of malignancy.

15. The method of claim 14, wherein said biological specimen is blood.

16. The method of claim 15, wherein after said biological specimen obtained, it is contacted with an agent capable of stabilizing said biological specimen.

17. The method of claim 14, wherein said magnetic particles are colloidal.

18. The method of claim 14, wherein after the step of preparing said magnetically-labeled sample, said sample is subjected to a high gradient magnetic field to produce a separated magnetically-labeled fraction which is enriched for said intact malignant cells, and said cell fragments or said cellular debris.

19. The method of claim 14, wherein said analysis is selected from the group consisting of:

multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, and automated cell analysis.

20. The method of claim 14, wherein said analysis further comprises classifying cell fragments or said cellular debris based on their origin as caused by apoptosis or necrosis.

21. The method of claim 20, wherein analysis further comprises classifying cell fragments or said cellular debris based on their origin as caused by mechanical damage, drug-induced damage, or immunological damage.

22. The method of claim 20, wherein said classification is based on at least one of the group consisting of:

morphologic analysis and epitopic analysis.

23. A method for diagnosing malignancy in a test subject comprising:

a. obtaining a biological specimen from a test subject, said specimen comprising a mixed cell population suspected of containing intact malignant cells and clusters of malignant cells;

b. preparing a magnetically-labeled sample wherein said biological sample is mixed with magnetic particles coupled to a first biospecific ligand which reacts specifically with said intact malignant cells and said clusters of malignant cells, to the substantial exclusion of other specimen components;

c. contacting said magnetically-labeled sample with at least one additional biospecific ligand which specifically labels said intact malignant cells and said clusters of malignant cells, to the substantial exclusion of other specimen components;

d. analyzing said labeled malignant cells and said labeled clusters of malignant cells, the presence of said labeled malignant cells and said labeled clusters of malignant cells indicating the presence of malignancy.

24. The method of claim 23, wherein said biological specimen is blood.

25. The method of claim 24, wherein after said biological specimen obtained, it is contacted with an agent capable of stabilizing said biological specimen.

26. The method of claim 23, wherein said magnetic particles are colloidal.

27. The method of claim 23, wherein after the step of preparing said magnetically-labeled sample, said sample is subjected to a high gradient magnetic field to produce a separated magnetically-labeled fraction which is enriched for said intact malignant cells and said clusters of malignant cells.

28. The method of claim 23, wherein said analysis is selected from the group consisting of:

multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, spectral imaging analysis, manual cell analysis, and automated cell analysis.

29. A kit for assaying a biological specimen for the presence of malignant cells, and cell fragments derived from malignant cells or cellular debris derived from malignant cells, comprising:

a. coated magnetic nanoparticles comprising;

i. a magnetic core material,

ii. a protein base coating material, and

iii. an antibody that binds specifically to a first characteristic determinant of said malignant cell, and said cell fragments or said cellular debris, wherein said antibody is coupled to said base coating material;

b. at least one antibody having binding specificity for a second characteristic determinant of said malignant cell, and said cell fragments or said cellular debris;

c. an agent capable of staining further features of said malignant cells, and said cell fragments or said cellular debris.

30. The kit of claim 59, further comprising a panel of antibodies each specific for a different characteristic determinant.

31. The kit of claim 59, further comprising a specific agent capable of labeling non-target entities.