WO2001006254A1

WO2001006254A1 - Cell enrichment

Info

Publication number: WO2001006254A1
Application number: PCT/US2000/019669
Authority: WO
Inventors: Blaise Bossy
Original assignee: Chromavision Medical Systems, Inc.
Priority date: 1999-07-19
Filing date: 2000-07-19
Publication date: 2001-01-25
Also published as: EP1210597A4; AU6111900A; EP1210597A1

Abstract

Provided are methods, compositions and kits for concentrating a desired cell type present in a biological sample. The method includes contacting a sample with a binding agent, wherein the binding agent is bound to a magnetic carrier molecule. The binding agent interacts with the desired cell type, which can thus be separated from the sample by use of the magnetic molecule.

Description

CELL ENRICHMENT

CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S.

Provisional Application Serial No. 60/144,529, filed July 19, 1999, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention generally relates to cell proliferative disorders and more particularly to enriching cells having a cell proliferative disorder in a biological sample.

BACKGROUND

Cell proliferative disorders can be characterized by a number of cellular changes, including expression of growth factors, growth factor receptors, adhesion molecules, and other cellular determinants which are readily identifiable to those of skill in the art.

The detection of cell proliferative disorders is important in detecting, diagnosing and treating neoplasms, and cancers. The detection limits of many assays are not sufficient to detect cells having proliferative disorders because the number of cells present in a sample are too few to provide a detectable signal.

Accordingly, there is a desire to increase the signal in order to adequately determine the presence or type of a cell proliferative disorder in a subject.

SUMMARY

The present invention provides methods and compositions useful in enriching a desired cell type contained in a sample. Such cells include, for example, those that can be characterized as having a cell proliferative disorder.

In one embodiment, the invention provides a method for enriching the number of neoplastic cells from a biological sample. The method is applicable to any number of cell proliferative disorders including, for example, neoplasms, and cancer. For example, the method of the invention can be used to enrich carcinoma cells from peripheral blood. In one embodiment, the invention provides a method of obtaining a composition substantially enriched in cells having a cell proliferative disorder, by obtaining a source of cells; contacting the source with a binding agent specific for an cell surface marker indicative of a cell proliferative disorder wherein the binding agent is bound to a magnetic bead and wherein the binding agent binds to cells in the source expressing the cell surface marker; and separating cells bound by the binding agent from the source thereby obtaining a sub-population of cells having a cell proliferative disorder. The binding agent is typically an antibody (e.g., monoclonal or polyclonal antibodies) . The method encompasses both positive and negative selection processes.

In another embodiment, the invention provides a method of obtaining a composition substantially enriched in cells having a cell proliferative disorder, by obtaining a source of cells; contacting the source with a binding agent specific for a cell surface marker indicative of a cell proliferative disorder wherein the binding agent is bound to a magnetic bead and wherein the binding agent binds to the cells expressing the cell surface marker in the source; separating the cells that are bound by the binding agent from the source thereby obtaining a sub-population of cells having a cell proliferative disorder; contacting the source with a second binding agent specific for a cell surface marker indicative of cells in the source which are not affected by a cell proliferative disorder, such that the second binding agent binds to the cells in the source; separating the cells that are bound by the second binding agent from the source thereby obtaining a second sub- population of cells having a cell proliferative disorder.

DETAILED DESCRIPTION

Mutation is the process whereby changes occur in the quantity or structure of the genetic material of an organism. Mutations are permanent alterations in the genetic material which may lead to changes in phenotype . Mutations can involve modifications of the nucleotide sequence of a single gene, blocks of genes or whole chromosomes. Changes in single genes may be the consequence of point mutations, which involve the removal, addition, or substitution of a single nucleotide base within a DNA sequence, or they can be the consequence of changes involving the insertion or deletion of large number of nucleotides. Modifications of whole chromosomes include both changes in number or structural changes involving chromosome abnormalities. Numerical chromosome mutations can involve multiples of the complete karyotype, termed "polyploidy, " or they may involve deviations from the normal number of chromosomes, termed "aneuploidy. " Mutations can arise spontaneously as a result of events such as errors in the fidelity of DNA replication or the movement of transposable genetic elements within genomes. They are also induced following exposure to chemical or physical mutagens . Such mutation-inducing agents include ionizing radiations, ultraviolet light and a diverse range of chemicals such as the alkylating agents, and polycyclic aromatic hydrocarbons, all of which are capable of interacting either directly or indirectly (generally following some metabolic biotransformations) with nucleic acids. The DNA lesions induced by such environmental agents may lead to modifications of the base sequence when the affected DNA is replicated or repaired and thus to a mutation. An increasing body of evidence implicates somatic mutations as causally important in the induction of human cancers. These somatic mutations may accumulate in the genomes of previously normal cells, some of which may then demonstrate the phenotypes associated with malignant growth. Such oncogenic mutations may include a number of different types of alterations in DNA structure, including deletions, translocations, and single nucleotide alterations. The latter, also known as point mutations, may frequently intervene in carcinogenesis, since a variety of mutagenic chemicals induce such mutations. In addition, such mutations may occur spontaneously as a result of mistakes in DNA replication. As used herein the term "mutant or mutated" as applied to a target neoplastic nucleotide sequence shall be understood to encompass a mutation, a restriction fragment length polymorphism, a nucleic acid deletion, or a nucleic acid substitution. A point mutation constitutes a single base change in a DNA strand, for example a G residue altered to a T. Such a mutation may alter the identity of the codon in which it lies thereby creating a missense mutation or nonsense mutation. Transition mutations involve the substitution of one purine in the DNA by another purine or one pyrimidine by another pyrimidine, that is A by G or vice versa, or T by C and vice versa. Transversions involve the replacement of a purine by a pyrimidine and vice versa. A missense mutation is a point mutation in which a codon is changed into one encoding amino acid other than that normally found at a particular position. A nonsense mutation is any mutation that converts a codon specifying an amino acid into one coding for termination of translation. Such nonsense changes are usually accompanied by the loss of function of the gene product. However, regardless of the type of change, a change in the amino acid sequence is potentially detectable by antibodies, such as monoclonal antibodies developed against a particular peptide sequence. A splicing mutation is any mutation affecting gene expression by affecting correct RNA splicing. Splicing mutations may be due to mutations at intron-exon boundaries which alter splice sites. A polyadenylation site mutant is a mutation of the consensus sequence required for addition of poly (A) to the 3 ' end of mature mRNA and which results in premature mRNA degradation. An insertion is any mutation caused by the insertion of a nucleotide or stretch of nucleotides into a gene. For example, naturally occurring insertion mutations can be the result of the transposition of transposable genetic elements.

Mutations that occur in somatic cells are not transmitted to the sexually produced offspring. However, such somatic mutations may be transferred to descendant daughter cells and mutations in some genes have been implicated in cancer. It is now clear that mutations may lead to the induction of cancer when they occur in one or more of a battery of normal genes referred to as the proto-oncogenes . Proto-oncogenes may be modified by a variety of mutational changes to produce the cancer- causing oncogenes. Proto-oncogenes play an essential part in the control of cell growth and differentiation and disruption of their normal activity by mutational events may lead to the aberrant growth characteristics observed in cancer cells.

The term "cancer," encompasses any carcinoma in a tissue of a subject. Such carcinomas would include, for example, carcinoma of the mouth, esophagus, throat, larynx, thyroid gland, tongue, lips, salivary glands, nose, paranasal sinuses, nasopharynx, superior nasal valut and sinus tumors, esthesioneuroblastoma, squamous call cancer, malignant melanoma, sinonasal undifferentiated carcinoma (SNUC) , or blood neoplasia.

Also included are carcinomas of the regional lymph nodes, including cervical lymph nodes, prelaryngeal lymph nodes, pulmonary juxtaesophageal lymph nodes, and submandibular lymph nodes. Other carcinomas include carcinomas of the breast tissue or ducts. By subject is meant any mammal such as bovine, canine, feline, porcine and humans.

Treatment of cell proliferative disorders such as neoplasms and cancer are becoming more common with the development of molecular biology and the understanding of cell cycle regulation. However, it is important to be able to diagnose a cancerous condition as early in its development as possible. It would be beneficial to be able to diagnose a cell proliferative disorder when the cells having the disorder at not so numerous as they become more difficult to treat and have a greater opportunity to metastasize.

The invention allows for diagnosis of cell proliferative disorders in biological samples containing a small percentage of cells having a disorder compared to the total number of cell in the sample (i.e., rare cells) . The invention provides a method whereby cells in a sample eliciting markers of a cell proliferative disorder can be efficiently concentrated from total cell content of the sample. Accordingly, by identifying and concentrating these "rare" cells the invention provides the ability to more accurately diagnose a cell proliferative disorder in a subject from a small sample or in samples where cancerous cells are rare. The invention provides methods, compositions, and kits that use antibodies (as described more fully below) , which recognize makers on cells indicative of a cell proliferative disorder. These antibodies are capable of binding to markers on a cell having a cell proliferative disorder. The antibodies themselves are bound to magnetic beads that are then used to separate the antibody-bound cells to concentrate them from the sample (e.g., by creating a "sub-sample"). Monoclonal Antibodies

Kohler and Milstein are generally credited with having devised the techniques that successfully resulted in the formation of the first monoclonal antibody- producing hybridomas (G. Kohler and C. Milstein, Nature, 256:495-497 (1975); Eur. J. Immunol., 6:511-519 (1976)). By fusing antibody-forming cells (spleen lymphocytes) with myeloma cells (malignant cells of bone marrow primary tumors) they created a hybrid cell line, arising from a single fused cell hybrid (called a hybridoma or clone) which had inherited certain characteristics of both the lymphocytes and myeloma cell lines. Like the lymphocytes (taken from animals primed with sheep red blood cells as antigen) , the hybridomas secreted a single type of immunoglobulin specific to the antigen; moreover, like the myeloma cells, the hybrid cells had the potential for indefinite cell division. The combination of these two features offered distinct advantages over conventional antisera. Whereas, antisera derived from vaccinated animals are variable mixture of polyclonal antibodies which never can be reproduced identically, monoclonal antibodies are highly specific immunoglobulins of single type. The single type of immunoglobulin secreted by a hybridoma is specific to one and only one antigenic determinant, or epitope, on the antigen, a complex molecule having a multiplicity of antigenic determinants. For instance, if the antigen is a protein, an antigenic determinant may be one of the many peptide sequences, generally 6-7 or more amino acids in length (M. Z. Atassi, Molec. Cell. Biochem., 32:21-43 (1980)), within the entire protein molecule. Hence, monoclonal antibodies raised against a single antigen may be distinct from each other, depending on the determinant that induced their formation; but for any given clone, all of the antibodies it produces are identical. Furthermore, the hybridoma cell line can be reproduced indefinitely, is easily propagated in vi tro or in vivo, and yields monoclonal antibodies in extremely high concentration.

Application of Monoclonal Antibodies to Cancer

Monoclonal antibodies are presently being applied by investigators to the diagnosis and treatment of cancer (for a general discussion of the topic, see Hybridomas in Cancer Diagnosis and Treatment, Mitchell, M. S. and Oettgen, H. F., (eds.), Progress in Cancer Research and Therapy, Vol. 21, Raven Press, New York (1982)). Monoclonal antibodies have been raised against tumor cells (U.S. Pat. No. 4,196,265), carcinoembryonic antigen (U.S. Pat. No. 4,349,528), and thymocytes, prothymocytes, monocytes, and suppressor T cells (U.S. Pat. Nos. 4,364,933; 4,364,935; 4,364,934; 4,364,936; 4,364,937; and 4,364,932). Recent reports have demonstrated the production of monoclonal antibodies with various degrees of specificity to several human malignancies, including mammary tumor cells (Colcher, D. et al . _r Proc. Natl . Acad. Sci. U.S.A., 78:3199-3203

(1981)), lung cancers (Cuttitta, F. et al . , Proc. Natl. Acad. Sci: U.S.A., 78:495-4595 (1981)), malignant melanoma (Dippold, W. G. et al . , Proc. Natl. Acad. Sci. U.S.A., 77:6114-6118 (1980)), colorectal carcinoma

(Herlyn, M. et al . , Proc. Natl. Acad. Sci. U.S.A., 76:1438-1442 (1979)), lymphoma (Nadler, L. M. et al . , J . Immunol., 125:570-577 (1980)), and neuroectodermal tumors

(Wikstrand, C. J. and Bigner, D. C, Cancer Res., 43:267- 275 (1982) ) .

Monoclonal Antibodies to Mammary Cells Several investigators have reported on the production of monoclonal antibodies against epitopes of various normal and malignant mammary cell components.

(Arklie, J. et al . , Int. J. Cancer, 28:23-29 (1981);

Ciocca, D.R. et al . , Cancer Res., 42;4256-4258 (1982); Colcher, D. et al . , Proc. Natl. Acad. Sci. U.S.A., 78:3199-3203 (1981); Foster, C. S., et al . , Virchows Arch. Pathol. Anat., 394:279-293 (1982); Greene, G. L. et al . , Proc. Natl. Acad. Sci. U.S.A., 77:5115-5119 (1980); McGee, JO'D. et al . , Lancet, 2:7-11(1982); Nuti, M. et al . , Int. J. Cancer, 291:539-545 (1982); and

Taylor-Papadimitriou, J. et al . , Int. J. Cancer, 28:17- 21(1981)). Many of the antigens recognized above are differentiation-related; therefore, these antibodies are most suited to histologically assess the differentiated status or grade of tumor specimens. For example, monoclonal antibodies directed against several antigens of human milk-fat-globule membranes have been produced. These antibodies have proven useful in studying the derivation of cell cultures, in evaluating the phenotypic expression of antigens in neoplastic transformation, have served as differentiation markers in breast cancer, and as immunodiagnostic reagents in the quantitation of antigens in the sera of breast cancer patients (Arklie, J. et al . , Int. J. Cancer, 28:23-29(1981); Ceriani, R. L. et al . , Proc. Natl. Acad. Sci. U.S.A., 74:582-586(1977); Ceriani, R. L. et al . , Proc. Natl. Acad. Sci., 79:5420- 5424 (1982); Foster, C. S. et al . , Virchows Arch. Pathol. Anat., 394:279-293(1982); and Taylor-Papadimitriou, J. et al . , Int. J. Cancer, 28:17-21(1981)). Arklie et al . have described monoclonal antibodies directed against human milk-fat-globule membranes. These antibodies showed a stronger staining reaction with well- differentiated (grade I) ductal carcinomas than undifferentiated (grade III) tumors. Nuti et al . have produced monoclonal antibodies against human metastatic breast carcinoma cells, which have been used to indicate tumor antigen heterogeneity. Foster et al . have also reported the production of monoclonal antibodies, which were used to show significant heterogeneity of antigen expression within breast tumors. Other reported monoclonal antibodies directed against carcinoma- associated antigens are identifiable by those of skill in the art . The invention provides methods and compositions for enriching cancer cells in a sample. One method employs positive selection and utilizes the binding affinity of antibodies directed to cell surface markers indicative of a cancer phenotype to purify these cells from non-cancer cells. Such techniques may employ column fractionation or affinity purification protocols.

An alternative carcinoma cell enrichment method, named negative selection, is based on the depletion of non-tumor cells present in a sample. This method utilizes antibodies directed to one or several cell surface markers expressed by non-carcinoma cells, such as CD45 expressed by white blood cells. The negative selection method offers the advantage of not relying on the presence of a carcinoma cell surface marker. These markers can have a wide range of expression due to the diversity of tumor cell prototypes .

Using the techniques and compositions described generally above, the Applicant has developed a method of enriching the number of neoplastic cells in a sample, using both positive and negative selection sequentially, to maximize the sensitivity of the carcinoma cell detection. Alternatively, each method (positive and negative selections) can be used alone. The method is described in the protocol outlined below, which is meant to illustrate, but not limit, the present invention.

EXAMPLES Example I: 20 ml of peripheral blood was drawn and anticoagulated with EDTA. The red blood cells were lysed for 5 minutes at room temperature with a red blood cell lysis buffer at a final concentration of 155 mM NH₄C1, 10 mK KHC0₃, 0.1 mM EDTA, at pH 7.2. Whole cells were separated from lysed red blood cells by centrifugation at 300 RCF for 5 minutes at room temperature. The supernatant was carefully aspirated and the RBC lysis step was repeated a second time with fresh lysis buffer. The supernatant was carefully aspirated again and the pellet was washed in PEB (PBS, EDTA, BSA; 1 x PBS, 0.1 mM EDTA, and 0.5% BSA) with an additional 5 minutes centrifugation tube. A 5 μl aliquot of the cell suspension was kept separately in a microcentrifuge tube to be added later in the cytospin of the positively selected cells to provide a minimum amount of cells at the end of this procedure.

Carcinoma cells expressing the human Epithelial Antigen (recognized by the monoclonal antibody HAE125) were enriched with magnetic beads by adding 0.1 ml of HAE125-microbeads (Miltenyi Biotec) to the 0.9 ml of cell suspension in the 1.5 ml microcentrifuge tube and incubated for 30 minutes at room temperature on an orbital shaker or a rotisserie.

An LS+ column on a midiMACS magnet (Miltenyi Biotec) was mounted and prepared with 3 ml of PEB. The cell suspension was loaded on the column, followed by 2 ml of PEB and 4 ml of PBS. The flow through was collected in a tube for the negative selection step. The column was eluted by removing the midiMACS magnet from the column and placing the column over a large capacity cytospin chamber (Hettich #1666) . 3 ml of PBS buffer was added to the column and collected in the chamber by gravity elution. A second 3 ml volume of PBS was added to the column and eluted by positive pressure (i.e., gently pushed through) . The eluant was collected in the chamber and mixed with the 5 μl aliquot of cell suspension taken before the positive selection. The cells from the eluant and the aliquot were spun together onto a slide in a cytocentrifuge at 500 RPM with a Hettich Universal 16A centrifuge (RevPro) for 15 minutes at room temperature. The slides were removed and allowed to dry for at least 1 hour at room temperature. The cell/magnetic bead ratio of the negative selection step can be optimized by one skilled in the art. In addition, the total cell number should not exceed the capacity (100 million cells) of the column used in the negative selection.

Based on the WBC titer determined in the sample of peripheral blood, the number of WBC originally present in the sample was calculated. Using this number, the volume of the flow through containing 100 million WBC was then determined and transferred to a separate tube and spun at 300 RCF for 5 minutes. The cells in the pellet were resuspended in 0.9 ml PEB and transferred in a microcentrifuge tube. A 5 μl aliquot of the cell suspension was kept with 0.5 ml PEB in the tube that was used to collect the flow through from the negative selection. The purpose of this aliquot was to provide a minimum of amount of cells at the end of the procedure. A volume of 150 μl of CD45-microbeads (Miltenyi Biotec, Inc. ) was added to the microfuge tube containing 0.9 ml of cell suspension and incubated for 15 minutes at room temperature on an orbital shaker or rotisserie. A LS+ column (Miltenyi Biotec, Inc.) was mounted and washed with 3 μl PEB. After the wash, the tube containing the 5 μl aliquot was placed below the column. At the end of the 15 minute microbead-cell incubation, 5 μl PEB was loaded on the column. The cell-microbead mixture was immediately added to the PEB on the top of the column.

The microfuge tube that contained the cell suspension was washed with 1 ml PEB, which was added to the column. When the top of the column was empty, the column was washed with 5 ml PEB. The flow through was clear, indicating that the negative selection worked properly. The tube containing the flow through from the negative selection was spun at 300 RCF for 5 minutes. The cells from the pellet were resuspended in 1.5 ml PBS and spun onto slides at low speed (500 rpm=32 RCF) with a Hettich Universal 16A centrifuge for 15 minutes at room temperature .

After the cytospin, the slides were removed and allowed to dry for at least 1 hour at room temperature. The slides were fixed with 400 μl 0.5% formalin for 10 minutes at room temperature in a moist chamber and washed two times in PBS for 3 minutes each. Permeabilization was performed by using standard buffers in Coplin jars. Antibodies specifically recognizing cytokeratins were mixed and incubated with the slides for 45 minutes or more at room temperature. The slides were washed and stained with standard buffers and chromogens for 10 minutes at room temperature. The slides were washed once in PBS and once in deionized water. Counterstaining with hematoxylin (DAKO #S3309) was performed for 4 seconds in concentrated stock solution, or for 20 seconds in a 5x diluted solution. The slides were then washed, incubated 30 seconds in a blueing solution (NH₄OH 0.08%), washed again in deionized water and dried at 70°C. The slides were finally mounted with a cellulose film (Sakura TissueTek SCA #4770) using xylene or, alternatively, mounted with Permount (Fisher #SP-15-100) and a 24x30 mm No. 1 glass coverslip (Fisher #125485G) . To enrich the carcinoma cells expressing the human Epithelial Antigen (recognized by the monoclonal antibody HAE125) , 20 ml of peripheral blood was drawn and anticoagulated with EDTA. The red blood cells were lysed for 5 minutes at room temperature with a red blood cell lysis buffer at a final concentration of 155 mM NH₄C1, 10 mM KHC0₃, 0.1 mM EDTA at pH 7.2. Whole cells were separated from the lysed RBCs by centrifugation at 300 RCF for 10 minutes at room temperature. The supernatant was carefully aspirated and the pellet resuspended in PEB (PBS, EDTA, BSA; lxPBS, 0.1 mM EDTA, and 0.5% BSA). The cell pellet was washed one time with an additional centrifugation and resuspension step as described above. The final pellet was resuspended in 0.9 ml of PEB and transferred to a 1.5 ml microcentrifuge tube.

Carcinoma cells were enriched with magnetic beads by adding 0.2 ml of HAE125-microbeads (Miltenyi Biotec Inc.) to the 0.9 ml of cell suspension in the 1.5 ml microcentrifuge tube and incubated for 30 minutes at room temperature on an orbital or rotary shaker.

An LS+ column on a midiMACS magnet (Miltenyi Biotec) was mounted and washed with 3 ml of PEB. The cell suspension was loaded on the column followed by 2 ml of PEB and 4 ml of PBS. The column was eluted by removing the midiMACS magnet from the column and placing the column over a Hettich #1666 chamber. 3 ml of PBS buffer was added to the column and collected in the chamber by gravity elution. A second 3 ml volume was added to the column and eluted by positive pressure (i.e., gently pushed through) . The eluant was collected in the chamber and spun onto slides in a cytocentrifuge at 500 RPM with a Hettich 16A centrifuge (RevPro) for 15 minutes at room temperature . The slides were removed and allowed to dry for at least 1 hour at room temperature. The slides were fixed in 400 μl 0.5% formalin for 10 minutes at room temperature in a moist chamber and washed 2x in PBS for 3 minutes each. Permeabilization was performed by using standard buffers in Coplin Jars. Antibodies were mixed and incubated with the slides for 45 minutes at room temperature. The slides were washed and stained with standard buffers for 10 to 15 minutes at room temperature. The slides were washed once in PBS and once in deionized water. Counterstaining with Mayer's hematoxylin (DAKO #S3309) was performed for 4 seconds in concentrated stock solution or 20 seconds in a 5x diluted solution. The slides were then washed, dried for 20 minutes at 70 °C, and mounted with an automated coverslipper (Sakura Tissue-Tek SCA), or manually with a glass coverslip and standard mounting medium, such as permount (Fisher #SP-15-100) .

Example II :

The following protocol is designed to enrich for carcinoma cells expressing the human Epithelial Antigen (recognized by the monoclonal antibody HAE125) , starting with 20 ml of peripherial blood containing EDTA. In order to keep a precise schedule, it is recommended not to use more than three samples simultaneously.

1. Red Blood Cells Lysis: a. Prepare lx lysis buffer (see stock solutions) out of a lOx stock solution with deionized or distilled water. We need 80 ml lx lysis buffer for each 20 ml blood sample. b. Prepare PEB (see stock solutions) . c. 20 ml fresh blood is equally split in 2 disposable 50 ml conical tubes (label tubes) . d. In each conical tube, add 40 ml lx lysis buffer mix by inverting the tubes; keep the tubes at room temperature for 5 minutes. e. Spin at 300 RCF for 10 minutes at room temperature. Remove the supernatant carefully by aspiration. f. Resuspend gently each pellet in 5 ml PEB by pepetting 3-5x and swirling. Pool the cells together into one conical tube. Wash the empty tube with 2 ml PEB, and add this to the other conical tube. g. Respin at 300 RCF for 5 minutes at room temperature. Remove the supernatant carefully by aspiration. h. Resuspend gently the cells in 0.9 ml PEB total as follows:

(i) the pellet's volumn is -200 μl; (ii) add first 500 μl PEB with a P1000

Pipetman to the pellett; (iii) resuspend the cells very gently by pepetting and swirling the cells; (iv) transfer the cells into a 1.5 ml Eppendorf tube; and

(v) rinse the bottom of the 50 ml conical tube with 200 μl PEB and add this to the Eppendorf tube (do not use small (P200) conical tips with the cells).

2. Carcinoma Cells Enrichment With Magnetic Beads: a. Add 0.1 ml HAE125 microbeads (Miltenyi Biotec

Inc.) to the 0.9 ml of cell suspension in the 1.5 ml Eppendorf tube . b. Incubate 30 minutes at room temperature on an orbital shaker or a rotisserie (such as the "Labquake" tube rotator from Barnstead/Thermolyne) . c. Assemble a LS+ column on a MidiMACS magnet (Miltenyi Biotec) , mounted on the black metallic rack. Make sure that the little wings of the column are in the front. d. Wash the LS+ column mounted on a MidiMACS magnet with 3 ml PEB. Put a 15 ml conical tube below the column to collect the flow through. e. Load the cells on the column. Wash the Eppendorf tube with 0.5 ml PEB and add it on the column. f. Wash the loaded column as follows:

(i) add 2 ml PEB and let it go through; (ii) add 2 ml PBS; and (iii) add 2 ml PBS again. g. Assemble a DAKO silanized slide with a carrier (Hettich #1670) , a large chamber (Hettich #1666) , and the corresponding ring for cytospin. Prepare a second chamber, if it is necessary to have a balancer during the cytospin, and fill it with 6 ml of water or

PBS. h . Elution :

(i) remove the column from the magnet and install it on a clip of the rotary shaker used as a rack (put the Hettich chamber below the column) ;

(ii) Add 3 ml PBS buffer on the column and let it drip by gravity into the assembled cytospin chamber; and (iii) Repeat this with 3 ml PBS and, in this case, push the buffer gently through the column with the corresponding plunger and collect it in the cytospin chamber (do not blow air through the column by pushing the plunger too far) .

3. Cytospin: a. Cytospin - the cells are spun at the lowest speed (-250 RMP) with a Hettich 16A centrifuge (RevPro) for 15 minutes at room temperature. b. Eliminate the supernatant with a vacuum pump linked to a Pasteur pipet having a disposable conical tip at its end. Avoid scratching the spot. c. Disassemble the chamber and dry the slid for at least 1 hour at room temperature. The slid is ready to be stained for sytokeratin.

ST CK S LUTI NS

1. lx Lysis Buffer: 155 mM NHRCl, 10 mM KHC03, 0.1 mM EDTA pH 7.2. lOx lysis stock solution for 1 liter: 82.9 g NH4C1 (SIGMA #A 0171); 10.0 g KHC03 (SIGMA #P 7682); 370 mg Na2 EDTA (SIGMA #E 5134) .

Check that the pH is 7.3 and store the stock solution at 4°C. Do not keep the lOx solution more than 1 month. Make the lx solution fresh daily with deionized or distilled H20.

2. PBS (Phosphate-Buffered Saline) :

PBS is either: 0.144 g/L KH2P04; 0.795 g/L NA2HP04-7H20; 9 g/L

NAC1; pH 7.4 (GibcoBRL #10010); or

20 mM sodium phosphate, 150 mM NaCl, pH 7.4

(DAKO S 3024) .

PEB (PBS, EDTA, BSA) :

PEB is lx PBS, 0.1 mM EDTA, 0.5% BSA.

For 50 ml mix:

0.25 g Bovine Serum Albumin, Fraction V (SIGMA #A 8022) ; 10 μl EDTA 0.5 M; and 50 ml PBS.

Vortex to dissolve the BSA. The PEB solution should be kept sterile or frozen. MAT RIAL

1. Clinical centrifuge with a swining rotor to spin 50 ml conical tubes at 300 RCF at room temperature.

2. Labquake tube rotator from Barnstead/Thermolyne (#400-110) .

3. Hettich Universal 16A centrifuge (#1601-1) from RevPro, with a rotor type #1387 with 6 swinging buckets (#1660) , the corresponding slide carriers

(#1670) with rings, and a sufficient number of disposable large chambers (#1666) .

4. MidiMACS starting kit (with LS+ columns and a

MidiMACS magnet) from Miltenyi Biotec (#423-01) .

5. HAE-125 Microbeads from Miltenyi Biotec (#611-01).

6. DAKO silanized slides (#S3003) . 7. 50 ml conical tubes, Eppendorf tubes, pipets, Pipetman, and conical tips.

R MARKS

1. It is desirable to use, for example, PBS (and not, for example, PEB) to wash the column before elution and during the elution steps . The presence of BSA would inhibit strongly the adherence of the cells to the slide.

2. It is important to centrifuge the cells without delay once they are loaded on the cytospin chamber. 3. The slide should be dried about 1 hour to overnight. Unstained slides should be conserved at -20°C in a dry environment.

4. Time Schedule: RBC lysis: 30 minutes;

L oedDD&ttME)EϋK.eHftat3ion : 30 minutes; Magnetic selection: 15 minutes; Cytospin: 15 minutes; Total: -1 hour, 30 minutes; Drying time: 1 hour minimum up to overnight; and Staining: 1 hour, 30 minutes to 2 hours.

After cytospin, the slide is dried for about 1 hour at room temperature. The spot containing the cytospun cells is circled with a hydrophobic pen

(such as DAKO Pen S2002), keeping the gap of -5 mm in between.

A. Fixation:

20x stock solution: 10% formalin, neutralized

(SIGMA #HT50-1-128) . lx fixation solution is made in PBS (0.5% formalin) just prior to the experiment.

Incubation: 400 μl 0.5% formalin for 10 minutes at room temperature in a moist chamber. Washes: 2x PBS for 3 minutes each.

B. Permeabilization: With solution A, diluted lOOx in PBS.

Incubation: 5 minutes at room temperature in a

Coplin Jar. Washes: 3x PBS for 3 minutes each. Antibody :

Mix 2.5 μl Ab (Solution C) /spot; 250 μl (Solution

D) /spot.

Incubation: 45 minutes at room temperature in a moist chamber. Washes: 3x PBS for 3 minutes each. (Negative control slides must be washed separately. )

D. Staining:

1.5 μl (Solution F)/spot; 0.6 μl (Solution G)/spot.

Mix these solutions and keep them in an open tube for 3 minutes at room temperature before adding: 300 μl (Solution E)/spot; 3 μl (Solution

H) /spot.

Distribute 300 μl/slide (or a bit less to make sure you have enough) .

Incubation: 10 minutes at room temperature. Washes: lx PBS for 3 minutes; lx deionized

H20 for 3 minutes.

E . Counterstaining :

With Mayer's hematoxylin (DAKO #S3309) . Incubation: 4 seconds in concentrated stock solution or 20 seconds in a 5x diluted (+dH20) solution. Washes: lx H20, up/down lOx in a beaker, then 2x deionized H20 for 3 minutes in a Coplin Jar.

The slides are then dried for 20 minutes at 70 °C and mounted. F. Mounting:

With a glass coverslip 24x30 mm No. 1 (such as Fisher #125485G) . The coverslip is kept away from the last 5 mm of the bottom of the slide to avoid the coverslip being squeezed by the ACIS carrier.

G. Mounting Media:

1. Permound (Fisher #SP15-100) . Press the coverslip gently to eliminate the surplus of mounting medium and to minimize the distance between the coverslip and the slid. This medium is permanent and does not need additional sealing. 2. As an alternative to a glass coverslip, the sample can be covered with a cellulose film from SAKURA Tissue-Tek automated coverslipper, using xylene . This method is permanent and does not introduce bubbles. This method can also be used manually.

Claims

WHAT IS CLAIMED:

1. A method of obtaining a composition substantially enriched in cells having a cell proliferative disorder, comprising: obtaining a source of cells; contacting the source with a binding agent specific for an cell surface marker indicative of a cell proliferative disorder wherein the binding agent is bound to a magnetic bead and wherein the binding agent binds to cells in the source expressing the cell surface marker; and separating cells bound by the binding agent from the source thereby obtaining a sub-population of cells having a cell proliferative disorder.

2. The method according to claim 1, wherein the binding agent is an antibody.

3. The method according to claim 1, wherein the sub- population is enriched for carcinoma cells.

4. The method of claim 1, wherein the selecting is done by positive selection.

5. The method of claim 1, wherein the selecting is done by negative selection.

6. The method of claim 2, wherein the antibody is monoclonal or polyclonal.

7. The method of claim 2, wherein the antibody recognizes an epithelial marker.

8. The method of claim 2, wherein the antibody is selected to avoid cross reactivity with the beads.

9. The method of claim 3, wherein the carcinoma cells are from peripheral blood.

10. A method of obtaining a composition substantially enriched in cells having a cell proliferative disorder, comprising: obtaining a source of cells; contacting the source with a binding agent specific for a cell surface marker indicative of a cell proliferative disorder wherein the binding agent is bound to a magnetic bead and wherein the binding agent binds to the cells expressing the cell surface marker in the source ; separating the cells that are bound by the binding agent from the source thereby obtaining a sub-population of cells having a cell proliferative disorder; contacting the source with a second binding agent specific for a cell surface marker indicative of cells in the source which are not affected by a cell proliferative disorder, such that the second binding agent binds to the cells in the source; separating the cells that are bound by the second binding agent from the source thereby obtaining a second sub-population of cells having a cell proliferative disorder.

11. The method according to claim 10, wherein the binding agent is an antibody.

12. The method according to claim 10, wherein the sub- population is enriched for carcinoma cells.

13. The method of claim 11, wherein the antibody is monoclonal or polyclonal.

14. The method of claim 11, wherein the antibody recognizes an epithelial marker.

15. The method of claim 11, wherein the antibody is selected to avoid cross reactivity with the beads.

16. The method of claim 10, wherein the carcinoma cells are from peripheral blood.