Provided are methods and compositions for isolating and detecting rare cells from a biological sample containing other types of cells, particularly including debulking that uses a microfabricated filter for filtering samples. The enriched rare cells can be used in a downstream process such as identification, characterization or growth in culture, or in other ways. Also included is a method of determining tumor aggressiveness or the number or proportion of cancer cells in the enriched sample by detecting telomerase activity, nucleic acid or expression after enrichment of rare cells. Also provided is an efficient, rapid method to specifically remove red and white blood cells from a biological sample containing at least one of the cell types, leading to enrichment of rare target cells including circulating tumor (CTC), stromal, mesenchymal, endothelial, fetal, stem, or non-hematopoietic cells et cetera from a blood sample.

Claims

43 · 3 independent · depth 7

43 granted claims

›1.independentA method of enriching target cells in a sample, which method comprises:na) Removing white blood cells (WBCs) from a biological sample by allowing them…+15

›2.↳ 1The method of claim 1, where the biological sample is a body fluid.d2

›3.↳ 1The method of claim 1, where the biological sample is blood or bone marrow or a tissue collected during surgery.d2

›4.↳ 1The method of claim 1, where the solid support comprises microparticles having an average size between 10 nm and 10 um.d2

›5.↳ 1The method of claim 1, where the solid support is linked to a specific binding member that binds to a ligand present on WBCs.d2+2

›8.↳ 1The method of claim 1, where the density-based approach involves centrifugation.d2+2

›9.↳ 1The method of claim 1, where the density-based approach uses a density medium comprising sugar or derivatives of sugar to establish a density gradient…d2+2

›12.↳ 1The method of claim 1, where removal of WBCs and removal of RBCs are performed at substantially the same time.d2

›13.↳ 1The method of claim 1 where the analysis, manipulation or application step comprises one of the following: flowcytometry, PCR, immunofluorescence, imm…d2

›14.↳ 1The method of claim 1 where the target cells are circulating tumor cells.d2

›15.independentA method for isolating a target cell from a biological sample containing other types of cells, said method comprising:na) enriching the sample by sele…+21

›16.↳ 15The method of claim 15, wherein the target cell is a cancerous cell.d2

›17.↳ 15The method of claim 15, wherein the biological sample is a blood sample.d2

›18.↳ 15The method of claim 15, wherein at least one type of non-target cell is removed by selective lysis.d2

›19.↳ 15The method of claim 15, wherein at least one type of non-target cell is removed by selectively adhering the non-target cell to a solid support.d2+6

›26.↳ 15The method of claim 15, wherein the presence, number, proportion, or property of target cell(s) in the sample is used to evaluate the subject from whi…d2+4

›30.↳ 15The method of claim 15, wherein the target cell is identified by a combination of two or more immunological staining processes.d2

›31.↳ 15The method of claim 15, wherein the target cell is identified by detection of telomerase activity.d2

›32.↳ 15The method of claim 15, wherein the target cell is identified by detection of a CD44 splice variant.d2

›34.↳ 15The method of claim 15, further comprising a step of determining whether the target cell has a therapeutic antibody bound to it.d2

›35.↳ 15The method of claim 15, which is used to evaluate a cancer therapeutic process or to determine a clinical end point for a cancer drug evaluation proce…d2

›36.↳ 15The method of claim 15, further comprising identifying at least one type of non-target cell in the sample and determining a ratio of target cells to a…d2

›37.independentA method for detecting a non-hematopoietic cancer cell in a blood sample, comprising the steps of:na) removing the majority of the hematopoietic cells…+4

Classifications

5 codes

IPC · International Patent Classification

Section C — Chemistry; metallurgy

C12Q1/24

USPC · US Patent Classification

435/30435/4436/177435/261

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Pendency

6.2 y

2,262 days filing → grant

Office actions

non-final + final

Responses

4 RCE

Examiner

Jim Ketter

art unit 1636 · TC 1600

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Priority chain

2 priority documents

Priority

14 Jul 2006

earliest claimed

›Priority documents — 2

Type	Document	Date
provisional	US 60831156	14 Jul 2006
related publication	US 20080206757 A1	28 Aug 2008

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