Methods and compositions for detecting non-hematopoietic cells from a blood sample

• US 20060252054A1
• Filed: 10/31/2005
• Published: 11/09/2006
• Est. Priority Date: 10/11/2001
• Status: Active Grant

• Current Assignee: AVIVA Biosciences Corporation
• Original Assignee: AVIVA Biosciences Corporation
• Inventors: Hujsak, Paul G., Snyder, Sara F., Yamanishi, Douglas T., Xu, Junguan, Voung, Elizabeth, Wang, Xiaobo, Wu, Lei, Cheng, Jing, Xu, Jia, Lin, Ping, Huang, Mingxian, Tao, Guoliang, Walker, George, O'Neill, Samantha, Schmitigal, Charina, Ouyang, Joe
• Granted Patent: US 8,980,568 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61B 5/150755 Blood sample preparation for further analysis, e.g. by separating blood components or by mixing
• CPC Class Codes: A61M 1/36 Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
• CPC Class Codes: A61M 1/3616 Batch-type treatment
• CPC Class Codes: A61M 1/3618 Magnetic separation
• CPC Class Codes: A61M 1/362 changing physical properties of target cells by binding them to added particles to facilitate their subsequent separation from other cells, e.g. immunoaffinity
• CPC Class Codes: A61M 1/3679 by absorption A61M1/3675 takes precedence
• CPC Class Codes: B01D 2325/0214 Tapered pores
• CPC Class Codes: B01D 57/02 by electrophoresis treatment of water, waste water, sewage or sludge by electrophoresis C02F1/469; electrophoretic production of compounds or non-metals C25B7/00; investigating or analysing materials by using electrophoresis G01N27/26
• CPC Class Codes: B01D 61/18 Apparatus therefor
• CPC Class Codes: B01D 67/0034 by micromachining techniques, e.g. using masking and etching steps, photolithography
• CPC Class Codes: B01D 67/0062 by micromachining techniques, e.g. using masking and etching steps, photolithography
• CPC Class Codes: B01D 71/027 Silicium oxide
• CPC Class Codes: B01D 71/04 Glass
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0457 : passive flow or gravitation
• CPC Class Codes: B01L 2400/0478 : pistons
• CPC Class Codes: B01L 2400/0644 : rotary valves
• CPC Class Codes: B01L 2400/065 : sliding valves
• CPC Class Codes: B01L 3/502 : with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: B01L 3/5027 : by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502753 : characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6841 : In situ hybridisation
• CPC Class Codes: C12Q 1/6881 : for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: G01N 1/34 : Purifying; Cleaning processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: G01N 1/40 : Concentrating samples
• CPC Class Codes: G01N 1/4077 : by other techniques involving separation of suspended solids
• CPC Class Codes: G01N 15/0272 : with screening; with classification by filtering B01D takes precedence
• CPC Class Codes: G01N 15/05 : in blood
• CPC Class Codes: G01N 2001/4016 : being a selective membrane, e.g. dialysis or osmosis
• CPC Class Codes: G01N 2001/4083 : sedimentation
• CPC Class Codes: G01N 2001/4088 : filtration
• CPC Class Codes: G01N 2015/012 : Red blood cells
• CPC Class Codes: G01N 2015/0288 : Sorting the particles
• CPC Class Codes: G01N 2015/0294 : Particle shape
• CPC Class Codes: G01N 2015/0681 : Purposely modifying particles, e.g. humidifying for growing
• CPC Class Codes: G01N 2035/0406 : Individual bottles or tubes
• CPC Class Codes: G01N 2333/4724 : Lectins
• CPC Class Codes: G01N 33/56966 : Animal cells
• CPC Class Codes: G01N 33/57407 : Specifically defined cancers
• CPC Class Codes: G01N 35/026 : having blocks or racks of reaction cells or cuvettes
• CPC Class Codes: G01N 35/04 : Details of the conveyor system G01N35/021 - G01N35/028 take precedence
• CPC Class Codes: G01N 35/1002 : Reagent dispensers
• 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 present invention recognizes that diagnosis and prognosis of many conditions can depend on the enrichment of rare cells, especially tumor cells, from a complex fluid sample such as a blood sample. In particular, the present invention is directed to methods and compositions for detecting a non-hematopoietic cell, e.g., a non-hematopoietic tumor cell, in a blood sample via, inter alia, removing red blood cells (RBCs) from a blood sample using a non-centrifugation procedure, removing white blood cells (WBCs) from said blood sample to enrich a non-hematopoietic cell, if any, from said blood sample; and assessing the presence, absence and/or amount of said enriched non-hematopoietic cell.

Claims

1. A method for detecting a non-hematopoietic cell in a blood sample, which method comprises:

a) providing a blood sample;

b) removing red blood cells (RBCs) from said blood sample, with the proviso that said RBCs are not removed from said blood sample via a Ficoll gradient centrifugation, and removing white blood cells (WBCs) from said blood sample to enrich a non-hematopoietic cell, if any, from said blood sample; and

c) assessing the presence, absence and/or amount of said enriched non-hematopoietic cell.

2. The method of claim 1, wherein the blood sample is a whole blood sample or a peripheral blood sample.

3. The method of claim 1, wherein the non-hematopoietic cell to be detected is a non-hematopoietic tumor cell.

4. The method of claim 3, wherein the non-hematopoietic tumor cell is a cancerous cell or a cancer cell.

5. The method of claim 1, wherein the RBCs are removed by sedimentation, filtration, selective lysis, or binding to a specific binding member that specifically binds RBCs

6. The method of claim 5, wherein the RBC sedimentation is promoted by specifically binding RBCs to a specific binding member that specifically binds RBCs to induce aggregation of the RBCs.

7. The method of claim 5, wherein the RBCs are removed by filtering the blood sample through a microfabricated filter comprising a tapered pore.

8. The method of claim 5, wherein the RBCs are selectively lysed by a hypotonic solution.

9. The method of claim 8, wherein the hypotonic solution comprises NH₄Cl, KHCO₃ and EDTA.

10. The method of claim 5, wherein the specific binding member that specifically binds RBCs is a lectin, a receptor ligand or an antibody.

11. The method of claim 1, wherein the WBCs are removed by binding to a specific binding member that specifically binds WBCs.

12. The method of claim 11, wherein the specific binding member is an antibody that specifically binds to WBCs.

13. The method of claim 12, wherein the antibody specifically binds to a ligand selected from the group consisting of CD3, CD11b, CD14, CD17, CD31, CD45, CD50, CD53, CD63, CD69, CD81, CD84, CD102 and CD166.

14. The method of claim 11, wherein the specific binding member is bound to a solid surface.

15. The method of claim 14, wherein the specific binding member is bound directly or indirectly to a solid surface.

16. The method of claim 14, wherein the solid surface is a magnetic particle.

17. The method of claim 1, wherein the RBCs are removed before the WBCs are removed from the blood sample.

18. The method of claim 1, wherein the WBCs are removed before the RBCs are removed from the blood sample.

19. The method of claim 1, which further comprises removing platelets or soluble proteins from the blood sample.

20. The method of claim 19, wherein the RBCs and the platelets are removed by treating the blood sample with a mixture of antibodies that facilitates the agglutination and precipitation of the RBCs and the platelets.

21. The method of claim 1, which further comprises debulking the blood sample.

22. The method of claim 1, wherein the presence, absence and/or amount of the enriched non-hematopoietic cell is assessed by labeling the enriched non-hematopoietic cell and identifying the labeled non-hematopoietic cell.

23. The method of claim 22, wherein the enriched non-hematopoietic cell is labeled by immunostaining, FISH, In situ PCR, In situ hybridization, or staining by a labeled binding member that specifically binds to the enriched non-hematopoietic cell.

24. The method of claim 22, wherein the labeled non-hematopoietic cell is identified by microscopic analysis, flocytometry or a laser based quantitative microscopy technology.

25. The method of claim 1, wherein the presence, absence and/or amount of the enriched non-hematopoietic cell is assessed by PCR.

26. The method of claim 25, wherein the PCR is singleplexed or multiplexed RT-PCR.

27. The method of claim 25, wherein the PCR is qRT-PCR or Real Time PCR.

28. The method of claim 1, wherein the presence, absence and/or amount of the enriched non-hematopoietic cell is assessed by assessing a non-hematopoitic marker in the enriched non-hematopoietic cell.

29. The method of claim 28, wherein the non-hematopoitic marker is an epithelial cell marker in the enriched non-hematopoietic cell.

30. The method of claim 29, wherein a plurality of the epithelial cell markers are assessed.

31. The method of claim 29, which further comprises assessing a tumor specific marker in the enriched non-hematopoietic cell.

32. The method of claim 31, wherein a plurality of the epithelial cell markers and/or the tumor specific markers are assessed.

33. The method of claim 1, which comprises subjecting the enriched, labeled and/or non-hematopoietic cell to additional analysis.

34. The method of claim 33, wherein the additional analysis is selected from the group consisting of PCR, oligonucleotide ligation assay (OLA), laser dissection microscopy (LDM), whole genome amplification (WGA), comparative genomic hybridization (CGH), DNA methylation assay, microarray analysis or a combination thereof.

35. The method of claim 33, wherein the additional analysis comprises assessing DNA methylation of the enriched non-hematopoietic cell.

36. The method of claim 27, wherein the non-hematopoitic marker is a protein or mRNA specifically expressed in epithelial and/or endothelial cells, DNA methylation pattern of the enriched non-hematopoietic cell or the chromosomal ploidy of the enriched non-hematopoietic cell.

37. The method of claim 3, wherein the tumor is a solid tumor.

38. The method of claim 37, wherein the solid tumor is selected from the group consisting of tumor of breast, colon, prostate, ovary, lung, liver, pancreatic, gastric, skin, melanoma, glioma, pharyngeal, esophagus, cervical and bladder.

39. The method of claim 1, which further comprises enriching the initially enriched non-hematopoietic cell.

40. The method of claim 39, wherein the initially enriched non-hematopoietic cell is further enriched by a positive selection of the non-hematopoietic cell or flowcytometry sorter.

41. The method of claim 22, which further comprises comparing the amount or number of the labeled and identified non-hematopoietic cell obtained in the test sample to the amount or number of the labeled and identified non-hematopoietic cell obtained in a comparable control sample.

42. The method of claim 25, which further comprises comparing the amount of a mRNA in the non-hematopoietic cell of the test sample to the amount of the mRNA in a non-hematopoietic cell obtained in a comparable control sample.

43. A method for detecting a non-hematopoietic cell in a blood sample, which method comprises:

a) providing a blood sample;

b) removing red blood cells (RBCs) from said blood sample by selectively lysing said RBCs, and removing white blood cells (WBCs) by specific binding of said WBCs to an anti-CD50 antibody from said blood sample to enrich a non-hematopoietic cell, if any, from said blood sample; and

c) assessing the presence, absence and/or amount of said enriched non-hematopoietic cell by;

i) immunostaining the enriched non-hematopoietic cell and counting the labeled cell;

ii) or PCR analysis of the enriched non-hematopoietic cell.

44. The method of claim 43, wherein the non-hematopoietic cell is a non-hematopoietic tumor cell.

45. A kit for detecting a non-hematopoietic cell in a blood sample, which kit comprises:

a) means for removing red blood cells (RBCs) from a blood sample, with the proviso that said means is not a Ficoll gradient centrifugation means, and means for removing white blood cells (WBCS) from said blood sample to enrich a non-hematopoietic cell, if any, from said blood sample; and

b) means for assessing the presence, absence and/or amount of said enriched non-hematopoietic cell.

46. The kit of claim 45, wherein the non-hematopoietic cell is a non-hematopoietic tumor cell.