Rare cell concentration

• US 10,444,240 B2
• Filed: 03/26/2014
• Issued: 10/15/2019
• Est. Priority Date: 03/29/2013
• Status: Active Grant

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Inventors: Pugia, Michael
• Primary Examiner(s): Claytor, Renee
• Assistant Examiner(s): Papciak, Sharon M.
• Application Number:
  US14/780,937
• Publication Number:
  US 20160054326A1
• Time in Patent Office: 2,029 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/574 for cancer
• CPC Class Codes: G01N 33/57488 involving compounds identifable in body fluids
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

A ratio of rare cells to non-rare cells in a blood sample suspected of containing rare cells and non-rare cells is enhanced. A treated blood sample is prepared by providing in combination the blood sample, a platelet deactivation agent, a fibrin-formation-arresting agent and fibrin in an amount sufficient to cause a predetermined level of agglutination of the rare cells. The treated blood sample is then contacted with a porous matrix such that agglutinated rare cells are preferentially retained on the porous matrix. The rare cells may then be identified.

Claims

1. A method of increasing a ratio of rare cells to non-rare cells in a blood sample suspected of containing rare cells and non-rare cells, the method comprising:

(a) adding in combination to the blood sample;

(i) a platelet deactivation agent,

(ii) a fibrin-formation-arresting agent, and

(iii) exogenous fibrin in an amount ranging from about 0.2 mg/L to about 10 mg/L to thereby yield a level of agglutination of the rare cells, the level of agglutination of the rare cells ranging from about 2.5% to about 100% based on an amount of surface area of the rare cells covered with large fibrin thereby preparing a treated blood sample comprising agglutinated rare cells; and

(b) contacting the treated blood sample with a porous matrix such that the agglutinated rare cells are preferentially retained on the porous matrix.

2. The method according to claim 1 wherein contacting the treated blood sample with a porous matrix comprises disposing the treated blood sample on a side of the porous matrix and applying pressure to the disposed treated blood sample.

3. The method according to claim 2 wherein the pressure applied is about 1 millibar to about 30 millibar.

4. The method according to claim 2 wherein the pore size of the porous matrix is about 1 μ

m to about 100 μ

M.

5. The method according to claim 1 wherein the rare cells are cancer cells.

6. The method according to claim 1 wherein prior to contacting the treated blood sample with the porous matrix the treated blood sample is diluted with a dilution medium.

7. The method according to claim 6 wherein the dilution medium is an aqueous buffered medium.

8. The method according to claim 1 wherein the agglutinated rare cells are subjected to an identification method.

9. The method according to claim 1 wherein steps (a) and (b) are performed simultaneously.

10. A method of increasing a ratio of cancer cells to non-cancer cells in a blood sample suspected of containing cancer cells and non-cancer cells, the method comprising:

(a) preparing a treated blood sample comprising agglutinated cancer cells by combining the blood sample with a combination comprising;

(i) a platelet deactivation agent,

(ii) a fibrin-formation-arresting agent, and

(iii) exogenously-added fibrin in an amount ranging from about 0.2 mg/L to about 10 mg/L to cause agglutination of the rare cells, the agglutination of the rare cells being in an amount of about 2.5% to about 100% based on an amount of surface area of the rare cells covered with large fibrin,

(b) diluting the treated blood sample with a dilution medium; and

(c) disposing the diluted treated blood sample on a side of a porous matrix and applying pressure to the disposed treated blood sample wherein the agglutinated cancer cells are preferentially retained on the porous matrix.

11. The method according to claim 10 wherein the pressure applied is about 1 millibar to about 30 millibar.

12. The method according to claim 10 wherein the pore size of the porous matrix is about 1 μ

m to about 100 μ

m.

13. The method according to claim 10 wherein the agglutinated cancer cells are subjected to an identification method.

14. A method of identifying at least one rare cell type in a blood sample suspected of containing the rare cell type, the method comprising:

(a) adding in combination to the blood sample;

a platelet deactivation agent,

a fibrin-formation-arresting agent, and

exogenous fibrin in an amount of about 0.2 mg/L to about 7.5 mg/L to cause agglutination of the rare cells, the agglutination of the rare cells ranging from about 2.5% to about 100% based on an area of an outer surface of the rare cells and to prepare a treated blood sample comprising agglutinated rare cells;

(c) diluting the treated blood sample with a dilution medium,

(d) contacting the diluted treated blood sample with a porous matrix such that the agglutinated rare cells are preferentially retained on the porous matrix; and

(e) contacting the agglutinated rare cells with an identification agent.

15. The method according to claim 14 wherein contacting the diluted treated blood sample with a porous matrix comprises disposing the treated blood sample on a side of the porous matrix having a pore size of about 1 μ

m to about 100 μ

m, and applying pressure to the disposed blood sample wherein the pressure applied is about 1 millibar to about 30 millibar.

16. The method according to claim 14 wherein the identification agent is a labeled specific binding member for the rare cells.

17. The method according to claim 14 wherein the rare cells are cancer cells.