Method for assessing disease states by profile analysis of isolated circulating endothelial cells

• US 20070154960A1
• Filed: 02/02/2007
• Published: 07/05/2007
• Est. Priority Date: 08/12/2005
• Status: Active Grant

• Current Assignee: Menarini Silicon Biosystems S.P.A
• Original Assignee: Veridex LLC (Johnson & Johnson)
• Inventors: Terstappen, Leon, Connelly, Mark, Doyle, Gerald, Rao, Galla
• Granted Patent: US 7,901,950 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: G01N 2800/32 Cardiovascular disorders
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent
• CPC Class Codes: Y10T 436/2525 Stabilizing or preserving
• CPC Class Codes: Y10T 436/25375 Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]

Abstract

Elevated number of Circulating Endothelial Cells (CEC) have been implicated in disease conditions associated with the formation or destruction of blood vessels such as acute coronary syndrome, thrombocytopenic purpura, sickle cell disease, sepsis, lupus, nephrotic syndromes, rejection of organ transplants, surgical trauma and cancer. This invention provides a method for assessing the levels of CEC which vary between different studies using a sensitive enrichment, imaging, and enumberation analysis. CD146 is one of the most specific endothelium-associated cell-surface antigens which can be used in image cytometry. CEC analysis provides an essential tool in prognostic/diagnostic evaluation in the clinic.

Claims

1. A method for assessing changes in cardiovascular homeostasis, comprising:

a. obtaining a biological specimen from a test subject suspected of having a cardiovascular disorder, a biological specimen comprising a mixed cell population suspected having target cells from the group consisting of endothelial cells, subpopulations of endothelial cells, epithelial cells, and combinations thereof;

b. contacting said biolgical specimen with a preservative capable of stabilizing said mixed population;

c. mixing said biological specimen with colloidal magnetic particles coupled to a monoclonal antibody which binds specifically to said target cells, to the substantial exclusion of other specimen components;

d. subjecting the specimen-magnetic particle mixture to a high gradient magnetic field to produce a separated cell fraction enriched in magnetic particle-bound target cells; and

e. analyzing said enriched fraction for the presence of target cells, the presence of said target cells is indicative of an change in homeostasis.

2. The method of claim 1, wherein said preservative contains an anti-coagulating agent and a stabilizing agent.

3. The method of claim 1, wherein said monoclonal antibody is from the group consisting of CD146, CD105, and combinations thereof.

4. The method of claim 1, wherein said analysis is an enumeration of target endothelial cells that are nucleated, CD146 positive, CD105 positive, and CD45 negative.

5. The method of claim 1 wherein said analysis is by a process seleced from the group consisting of image cytometery analysis, fluorescent in situ hybridization, and combinations thereof.

6. The method of claim 1 wherein said analysis is indicative of changes in stenosis, organ transplant status, heart disease, or combinations thereof.