MX2010013509A - Device and methods for the detection of cervical disease. - Google Patents

Abstract

The present invention discloses a novel device and methods for the detection of the cervical disease, including cervical dysplasia and cervical carcinoma (which ranges from carcinoma in situ to invasive carcinoma in the cervix), in several samples at the same time. The "multiwell" device of the present invention consists of a solid support featuring multiple well-separated areas, each accommodating a patient sample, leading to simultaneous evaluation of patient samples so as to be analysed and detecting the cervical disease in a screening using the method of the present invention.

Description

METHOD AND DEVICE FOR THE DETECTION OF CERVICAL DISEASE FIELD OF THE INVENTION The field of the invention is the detection of cervical disease and its screening, using a device that renews the methodology.

BACKGROUND OF THE INVENTION The present invention describes a device for new methods for the detection of cervical disease, including cervical dysplasia and cervical carcinoma (ranging from carcinoma in situ to invasive carcinoma of the cervix), in several patient samples at the same time.

The "multi-cell" device of the present invention consists of a solid support that has several areas of good separation, each serving to accommodate a sample of a patient, leading to simultaneous evaluation different samples of different patients, for review and detection of cervical disease in a screening, using the method of the present invention.

The methodology of the present invention, first comprises the conventional Cytological spots applied by the new "multi-cell" device, which includes the conventional hematoxylin or Hematoxylin and eosin staining of (H &E) and conventional Papanicolau staining.

Furthermore, the methodology of the present invention comprises determining the expression of at least one biomarker or the combination of some biomarkers in cervical cells collected directly from the cervix of a patient or derived from a cervical sample of a patient. Such cervical cells may be adhered to the walls of the "multi-cell" device or suspended in the cell.

In addition, said determination of expression may depend on the use of an antibody or the combination of antibodies.

In particular, the method of the present invention is described herein by an antibody, or a combination thereof. Said antibodies, or variants and fragments thereof, are capable of binding to biomarkers that are overexpressed in cancer, including cervical carcinoma and dysplasia, as compared to a normal control.

In addition to practicing the methodology of the invention the "multi-cell" device is presented in a kit.

Finally, the device and methodology of the present invention can be practiced with a manual mode method, or modified to a semiautomatic or fully automated mechanism according to the skills that are known. To the extent, the methodology of the present invention has its application in the innovative way of carrying out screening for detection and for the management of cervical disease.

It should be noted that the current invention covers the use and application of the device and methods to any biological fluid, or that the cell suspension is extracted from any biological fluid for sample for the detection and handling detection of other diseases and conditions relevant to others organs, beyond gynecological and cervical diseases.

Abbreviations: cervical cancer (CvC).

The incidence of cervical cancer and its mortality.

Cervical cancer occupies the second in the diagnosis of malignancy and the third place as a cause of death by cancer in women around the world. There are 555, 100 new cases and 309,800 deaths calculated in 2007, 83% of which have occurred in the developing world (American Cancer Society, ACS World Cancer facts and figures, 2007).

In the United States in 2010, there were an estimated 12,200 new cases of invasive cervical cancer and an estimated 4,210 deaths from cervical cancer (American Cancer Society, ACS Cancer Facts &Figures, 2010; Jemal A, et al., Cancer Statistics, 2010, CA Cancer J Clin 60: 277-300, 2010).

Since its inception in 1957, the Pap test has become a routine test for the detection of cervical-uterine disease in the United States. As a result of this routine successful screening test, the incidence rates of cervical cancer have decreased in the African-American female population and in Caucasian women, pre-invasive lesions of the cervix are detected much more frequently that invasive cancer, and mortality rates have declined so in recent decades. Etiology of cervical cancer and pathology. Infection with human papilloma virus (HPV) is a major etiological factor for the etiology of cervical cancer. The magnitude of the risk association is greater than smoking for lung cancer (Unger ER, Barr E, human papilloma virus and cervical cancer, in Emerg Infect Dis 10: 2031-2032, 2004). Among the known types of HPV 200, HPV16 / 18 are the strains most commonly associated with cervical cancer, more than a risk greater than 200 v eces than the rest of the strains. (Castellsague X, et al., 1A1 around the world has been considered the infection of human papillomavirus (HPV) and its co-factors as the etiology of cervical adenocarcinoma: Implications for detection and prevention, J Nati Cancer Inst 5: 303-315, 2006).

However, most HPV infections disappear spontaneously and only a small percentage progress to NCI (cervical intraepithelial neoplasia) or CIS (carcinoma in situ). Other risk factors that contribute to the production of cervical cancer disease can be immunosuppression, multiparity, the smoking habit, and nutritional factors, as well as the prolonged use of oral contraceptives (OS / Information Center). ICO in HPV and cervical cancer, summary report on HPV and cervical cancer statistics in Brazil, 2007, ACS, 2010).

Cervical cancer comprises two main types: squamous cell carcinoma (75%) and adenocarcinoma (20%), which affect the squamous cells and the glandular cells of the cervical epithelium respectively (WHO / ICO, 2007).

The Bethesda system classifies precancerous cervical lesions into: I) Atypical squamous cells of undetermined significance (ASCUS) II) LOW DEGREE INTRAEPITELIAL LESIONS (LSIL) or cervical intraepithelial neoplasia (NC - I), characterized by mild dysplasia.

III) High-grade intra-epithelial 1 squamous lesions (HSIL) or cervical intraepithelial neoplasia, including carcinoma in situ (NCI-II, NCI-III / CIS) characterized by moderate to severe dysplasia.

Note that LSIL / HSIL nomenclature refers to cervical lesions detected by the cytological test, while the NCI nomenclature refers to the dysplasia determined according to the histological analysis of the biopsy of the sample taken from cervical tissue (WHO / ICO , 2007).

CIN - I, rarely (1%) evolves to cancer and mostly returns to normal, even without any treatment; CIN - II carries a risk of progression in cancer of 16% for two years and 25% after five years, if no treatment is given. CIS (CARCINOMA IN SITU) is a cervical cancer confined to the uterine cervix that will evolve to invasive cervical cancer (CCI) in a period of between 10 and 12 years. Relative survival for patients with cervical cancer in the U.S.A. from 1 year to 5 years is 88% and 72% respectively, while the survival rate for patients diagnosed with 5-year-old cervical cancer is 92% (ACS, 2010).

Figure 1 illustrates the stages of cervical disease.

NCI precancerous lesions can be treated by electrocoagulation, cryotherapy, C02 laser ablation or local surgery (Campion M, Pre-invasive disease.

Published In: Gynecologic Oncology Practices Third Edition. Eds: Berek JS, hacker NF, Lippincott, Williams & Wilkins, Philadelphia, United States, pp 271-343, 2000; ACS, 2007). The different types of invasive cervical cancer are treated with surgery, radiation or both, as well as chemotherapy can be used in specific cases and with previous selection.

The symptoms are often abnormal vaginal bleeding, it does not occur until the cancer has developed.

Detection of cervical cancer.

Papanicolaou examination in developed countries has contributed to doubt, the decrease in the incidence of morbidity and mortality of cervical cancer, due to the early detection of cervical lesions. The Papanicolaou test is a cervical cell cytological staining collected through a simple procedure that is performed in the office. The cells are smeared either directly into a lamella, then fixed and stained this is the sample preparation for the classic and (conventional Papanicolaou), or first rinse in a liquid preservative solution to separate the cells from the mucosa and remove cell detritus from the sample, before preparing the lamellae (liquid based cytology, CBL). In both cases, the lamellae are read and reviewed by a pathologist.

The Papanicolaou Test is based on subjective visual reading of cell morphologies and its sensitivity has been limited to one (50%) and high susceptibility to a variability in the intra and interpersonal sample appreciation (Boulet GAV, et al., HPV human papilloma virus in the detection of cervical cancer: and the important role that biomarkers play, CANCER EPIDEMIOLOGÍA BIOMARCADORES PREV 17: 810 - 817, 2008).

They can be read from 50,000 to 300,000 cells per slide, in order to find potentially abnormal cells that are 20-30 cells, and a double reading is often required. Therefore the Pap test. of highly qualified and trained personnel in suitable laboratories, to make the tests that require a lot of work and are very expensive.

The development of CBL (LIQUID BASE CYTOLOGY) technology (ThinPrep, Cytyc Inc., Safe Path, Three Pathways for Image Processing, Inc.) has provided a more standardized method of sampling, thus reducing by > 50% the number of inadequate cell samples, while allowing the detection of more than 65% LSIL in the general population that performs the conventional Papanicolaou test. In addition, automated slide preparation and automated reading methods have reduced the burden of analysis, as well as intra- and interpersonal variability for the evaluation of the sample. However, to date, the Pap test even in its CBL format (LIQUID BASE CYTOLOGY) remains a "reading" based on screening for its detection, which requires an appropriate infrastructure and trained and trained human resources .

The etiological relationship between HPV and cervical cancer has been exploited for the development of molecular technologies for the identification of the virus and to be able to overcome the limitations of the cytological study in the cervical detection that is carried out in the screening. The HPV DNA Amplification Test is now used to compensate for mistakes in Pap tests, to classify high-risk patients, to address them and send them to a cytological test and as a follow-up after NCI treatment (Boulet, 2008) . Currently the HPV test serves as a substitute endpoint for screening and detection of cervical cancer. However, it has also been suggested for the primary examination, particularly in women over 30 years of age, followed by a screening cytology if they turn out to be HPV positive (Smith RA, Cokkinides V, Eyre HJ.) GUIDES FOR EARLY CANCER DETECTION, OF THE AMERICAN SOCIETY OF CANCER, 2005. CA Cancer J Clin 55:31 - 44, 2005). HPV tests by DNA amplification are performed in BASE LIQUID FOR CYTOLOGY (LBC) ThinPrep samples, such as Papanicolau staining.

In accordance with the recommendations of the SAC (Smith, 2005), the detection of cervical cancer must be done every year with conventional pap smears or on liquid basis for cytology (LBC). If the Pap test is abnormal, it reveals ASCUS, the HPV test is performed, and if it is positive, the women refer to colposcopy. If the Pap test reveals LSIL or HSIL, patients immediately refer to colposcopy.

Colposcopy is the microscopic examination of the cervix to acetic acid or Lugol's stain to reveal the abnormal cells, which can in turn be referred for biopsy. Women over 30 years who have had three normal Pap test results in a row, can have a review every 2-3 years with cervical cytology alone, or every 3 years with a HPV DNA test plus cytology cervical. Women older than 70 years who have had three or more normal PAP of consecutive tests and no Pap test was abnormal in the last 10 years, may stop screening tests (Smith, 2005).

Immuno-labeling can be used to improve the accuracy of the Papanicolaou test, complementing the reading with a more objective criterion basing the cytological test on the antigen-antibody interaction.

The Ki-67 antigen is a large nuclear protein, which is expressed in the proliferation of cells (Goodson WH., Et al.) The functional relationship between in vivo bromodeoxyuridine labeling index and proliferation index of Ki-67 in cancer. human breast, Breast Cancer Res Treat 1998 May; 49 (2): 155-164; Scholzen T., et al., Ki-67 protein: from the known and the unknown [review], J. Cell Physiol 2000; 182 : 311-22). Ki-67 is expressed preferentially during the active phases of the entire cell cycle (final G 1 -, S, G 2 - and M-), and is absent in resting cells (G 0 -). In anatomo-pathological diagnosis, antibodies to Ki-67 are used for tumor grade proliferation rates (Cattoretti g., Et al.) Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIBland MIB3) to detect cells that are proliferating in samples processed in paraffin fixed in formalin in microwaves J Pathol 1992; 168: 357-63).

Immunostaining of Ki-67 with the commercially available antibody MIB-1 has been evaluated as an adjunct test to improve the accuracy of the diagnosis of squamous intraepithelial cervical lesions (LSIL or HSIL; Pirog et al.

The accuracy of the diagnosis of low-grade squamous intraepithelial cervical lesions has been improved with immunostaining with MILB-1, Am J Surg Pathol 26: 70-75, 2002). In fact it was reported that there is considerable variation in the diagnosis of LSIL between observation of the sample and immunostaining.

Immunostaining with MIB-1 will be more sensitive and specific than HPV testing.

It has been demonstrated that mini-chromosomes of maintenance proteins (MCM) and in particular MCM-2, MCM-5 and 7 of MCM, are useful for the detection of cervical disease including Dysplasia and cancer (Williams et al. al., Proc Nati Acad Sci USA 95: 14932-14937, 1998; Freeman et al., 5: 2121 Clin Cancer RES-2132, 1999), as has been demonstrated in conventional cervical smears or by immunohistochemical staining of cervical tissues. Recent results using a mouse transgenic HPV model have shown that MCM-7 rather seems to be a marker of immunochemistry for a high quality detection of cervical disease (brake et al., Cancer res.) 63: 8173- 8180, 2003; Malinowski et al., Acta Cytol., 43: 696, 2004; 7, 632, 498, U.S. Patents, Malinowski et al., 2009).

Cyclin 2A inhibitor-dependent kinase (CDKN2A), also known as pl6 (INK4a) is a regulatory cell cycle of overexpression in preneoplastic cervical lesions harboring HPV16 / 18 and cervical cancer. The over-expression of p6 (INK4a) is due to the functional inactivation of the Rb retinoblastoma protein, by the HPV E7 protein. Therefore the over-expression of p6 (INK4a), is related to the active expression of HPV genes, and is preferable to only detect the presence of the virus.

Therefore it has been proposed that - overexpression of p6 (INK4a) can be used as a marker to detect persistent high-risk HPV infection and increase the quality of detection of squamous epithelial lesions (HSIL); Klaes et al. Over-expression of p6 (INK4a) and as a specific marker for dysplasias and neoplastic epithelial cells of the cervix, Int J cancer 92: 276-284, 2001; Agoff et al., Expression of pl6 (INK4a) correlates with some degree of cervical neoplasia: a comparison with Ki-67 expression and detection of high-risk HPV types, Mod Pathol 16: 665-673, 2003; Von Knebel Doeberitz et al., 2004, 6,709,832 of United States patents).

Detection by immunohistochemistry with the p6 (INK4a) in cervical biopsies is in routine use to discriminate between the association or not of the HPV lesions (Redman R, et al., The usefulness of P16 (Ink4a) in discriminating between neoplasia cervical intraepithelial 1 and non-neoplastic equivocal lesions of the cervix, 132: 795 Arch Pathol Lab Med-799, 2008) and as a substitute marker for high risk of HPV infection (ulvany NJ, et al., diagnostic utility of pl6lNK4a: a re-evaluation of its use in cervical biopsies, Pathology 40: 335-344, 2008).

In addition, immunostaining of cervical-uterine tissue samples with ThinPrep pl6lNK4a is being evaluated for its ability to aid in the identification of high-grade intraepithelial lesions, evaluated in follow-up biopsies and high-HPV DNA tests. risk (Meyer et al., evaluation of pl6 INK4a expression of ThinPrep cervical specimens with the pl6 CINtec INK4a trial, cancer, 111: 83-92, 2007; Doeberitz et al., 2007, 7,306,926 of US patents).

Finally, a procedure based on the ELISA test with pl6 (INK4a) for the detection using an exfoliative cell protein lysate, it has been reported that a greater sensitivity and specificity for the dysplasia and cancer tests (Ding L, et al. al., proof of ELISA to detect the CDK2A of the ThinPrep (expression of pl6 (INK4a) in the exfoliating cells: it is a new tool for the detection test of the cervico-uterine cancer, Mol Diagn Ther 12: 395-400, 2008).

In conclusion, the use of antibodies against specific biomarkers of cervical cancer and cervical pre-neoplastic lesions can increase the accuracy of detection of current cervical disease.

There is a consensus regarding discrepancies in interpretation that fall into error with common observers, especially in the LSIL cytology category. In fact immunization with antibodies represents an "objective test" therefore it can complement the morphological interpretation offered by the pathologist. Antibodies against specific biomarkers could help in the interpretation for the diagnosis of LSIL, by increasing the accuracy of anatomopathological diagnosis based on cytology.

Among the circulating biomarkers, serum levels of squamous cell carcinoma antigen have been found to be related to the stage of the tumor, the size of the tumor, the residual tumor after treatment, or recurrent tumor or progressive disease and thus the Survival in cervical squamous carcinoma (KN from Gaarenstroom, Bonfrer JMG, NACB, National Academy of Clinical Biochemistry: guidelines for the use of tumor markers for cervical cancer, 2007). SCC antigen is a group of glycoproteins with a molecular weight of ~ 45KDa, belonging to the family of serine protease inhibitors. There are in fact two genes, SSC1 and SSC2, both located on chromosome 18q21.3, for the coding of the neutral and acid isoforms, respectively. The neutral form is detected in malignant and normal epithelial cells.

Considering that the acid isoform is found in tumor cells and in the sera of patients with well-differentiated carcinoma of squamous cell carcinoma (Kato H, et al., Heterogeneous distribution of TA-4 acids in cervical squamous cell carcinoma; Immunohistochemistry with monoclonal antibodies, 78: 1246 Japanese J Cancer Res-1250, 1987). SSC1 and SSC2 are almost identical, only differ in their reactive handles. There is evidence that the events regulating proteolytic processes, in both normal processes and pathological processes, still have different biological functions (De Bruijn HA, et al., The clinical value of squamous cell carcinoma antigen in cancer of the neck uterine, Tumor Biol 19: 505-516, 1998).

Elevated levels of SCC have also been found in patients with squamous cell carcinoma in other organs (vulva, vagina, head and neck, lung) as well as in patients with benign skin diseases (psoriasis, eczema), lung (sarcoidosis), liver and kidney.

However, according to NACB recommendations (Gaarenstroom, 2007), SCC is not a biomarker sensitive enough to detect cervical cancer and its clinical usefulness in prognosis, but with respect to the monitoring of response to treatment more evaluation is needed.

In conclusion, there is a need to improve the accuracy, the development of the methodology and the normal cost in the detection of cervical disease. The device and methodology of the present invention have utility and should be applied in the detection of cervical disease as a novelty based on high performance cytology.

SUMMARY OF THE INVENTION: The present invention reveals a device and a novel methodology for the detection and screening of cervical disease in cervico-uterine samples of patients.

The "multi-cell" device of the present invention consists of a solid support with multiple well-spaced circular areas to place each sample of a patient, thus providing accommodation to multiple patient samples, thus enabling simultaneous evaluation of multiple patient samples. , for the detection and screening of cervical disease, using the methodology of the present invention.

The methodology of the present invention first comprises the conventional cytological spots that apply to the innovative device, cells or wells, including the conventional method of staining with hematoxylin or Hematoxylin and eosin (H & E) and conventional staining of the Papanicolau. Therefore, the device and method of the present invention has utility application in the detection of high performance, detection and management of cervical disease, as well as a variety of other cell-based and cytological assays.

The present invention encompasses the application of the device and the methodology of the present invention in patient samples that come from any fluid or biological or tissue. To the extent that the present invention provides useful applications in the analysis, detection, diagnosis, disease management and beyond detection of a variety of diseases and conditions of different sites and organs beyond gynecological diseases.

Pre-neoplastic, dysplastic and malignant (Thin Prep) cervical cells are rare.

Therefore, in the preferred field of the present invention, an experimental condition is established by copying a sample of cervical cancer patient from the screening, mixing the normal cervical cells with the cells of a cancer cell line, They grow together in co-culture. In this example of co-culture shown below, it was used to illustrate the methods of the present invention. The use of authentic Thin Preps, the most common preparation in cervical-uterine tissue samples that is currently used for screening and management of cervical cancer and others.

All forms of cervical samples are included in the present invention.

The present invention also provides a novel method of immunostaining cervical-uterine tissue samples from a patient with cervical disease using the "multicell device", or "pouch device". In one assay, the present invention was preferably used, and in the assay a mixture of normal cervical cells and cervical cancer cells was used to simulate a sample with cervical tissue, with a specific antibody, destined to locate a surface antigen located in the Cell membrane, are used to stain cancer cells between normal cervical cells. The present invention exploits the advantages of using specific monoclonal antibodies to identify some cancer cells in a mixture of normal cells, which increases diagnostic accuracy, on the visual evaluation of the morphology of the cell by the pathologist.

Also included in this invention is the use of an antibody or the combination of antibodies against a biomarker or a combination of biomarkers that are associated with pre-neoplastic, neoplastic or simply dysplasia in the cervical cells. The use of antibodies against said biomarkers would greatly improve the detection sensitivity of the current test.

Finally, the present invention details a novel method for the detection of cervical disease and that is practiced in a suspension of cells in a detection solution. The suspension of said cells is a mixture of normal cervical cells and cells with cervical cancer, as described above, an imitation of experimental condition a sample of the patient with cervical tissue for the detection of cervical disease. The authentic ThinPrep solution is comprised by the present invention. In this re-creation of the model, the staining of the cell is carried out with an anti-Her2 antibody, which is located in the cell membrane of the cancer cells of the model used. However, the antibody or the combination of antibodies against biomarkers or combination of biomarkers that are associated with preneoplastic, neoplastic lesions or cervical cell dysplasias are carried out by the present invention and would further greatly improve current PAP detection sensitivity.

The incorporation of the latter also offers the possibility of quantifying the specific antigen-antibody reaction, in turn facilitating and potentially automating the entire screening process for cervical cancer. In the present example a colorimetric reaction is used, but they are from other detection systems carried out also using the present invention.

Immunostaining based on colorimetric reading of cell reactivity represents an accurate, cost-effective and easy-to-use improvement with a visual review for the evaluation of staining intensities and cell morphologies performed by the pathologist.

In addition kits are provided to practice the methodology of the invention.

Persons skilled in the art affirm that in the present invention the devices and methodology are included to carry out or practice the procedure manually, or the methodology can be modified to perform the procedure semi-automatically, or it can also be performed in a completely automated In conclusion, the present invention offers many useful enhancements and applications for the most current cervical cancer screening procedures: Unique high performance to perform multiple procedures simultaneously Greater precision in the diagnostic interpretation, comparing the morphological evaluation of the cell, with the proper use of specific antibodies which makes it more profitable and prestigious due to greater specificity and sensitivity.

The quantitative measurement of the antigen-antibody reaction specifies.

BRIEF DESCRIPTION OF FIGURES AND TABLES Figure 1: Design of the cell-cell device, the multi-cell device of the present invention is shown. Prototype is 90 x 130 x 1 mm. , presenting a numbered (1-40) of the circular areas (or cells) of 15 mm in diameter. Other formats are also contained in the present invention, such as, but not limited to: 6, 12, 24, 48, 96, 384-cells or any other size, as long as the diameter is compatible with the performance of the test and with the device in general which can be easily mounted and read under a microscope.

Figure 2A: Staining of normal cervical cells with Hematoxylin, using the multicell device. Normal cervical patient samples, that is, 250 M 1 of a Thin Prep, circular areas of 15 mm diameter of the multi-cell device are deposited in each of the cells of the multi-cell device. Therefore, several samples can be evaluated simultaneously. The cells are air-dried in RT and rinsed with PBS. To eliminate excess waste from the cell, then the cells are ready for any cytological staining procedure. In the present document, hematoxylin staining is shown for four different samples (2A-2B) of the multi-cell device. The device is rinsed with ddH20, and the samples are covered with a few drops of weak Mayer hematoxylin solution for 1-2 minutes, then washed with blue nucleus bluing solution or H20 tap. The device is observed under a microscope with different magnifications (Figure 2A: 4 X; Figure 2B-2D: 10 X). The results show cytological staining that can be correctly interpreted in the multi-cell high performance device of the present invention.

In Figure 2?: Sample of Normal Cervical Cells that is processed in the multi-cell device fixed and stained with hematoxylin (4X magnification).

Figure 2B: Sample of Normal Cervical Cells that is processed in the multi-cell device fixed and stained with hematoxylin (10X magnification) Figure 2C: Sample of Normal Cervical Cells that is processed in the multi-cell device fixed and stained with hematoxylin (10X magnification) Figure 2D: Sample of Normal Cervical Cells that is processed in the multi-cell device fixed and stained with hematoxylin (10X magnification) Figure 3A: Immunostaining of a sample containing a mixture of cells with cervical cancer and normal cervical cells, using the multi-cell device and antibodies commonly used. The normal cervical cells come from a sample of a patient without cancer or is normal from a liquid-based preparation (Thin Prep) and mixed with BT474 breast cancer cells in an experiment where a co-culture is performed imitating a sample of a patient for detection of cervical cancer, as described in examples 2 and 3, with the methodology used in the present invention together with the multi-cell device. Then, an immunostaining with specific antibodies is performed.

Specifically, we have a constant amount of normal cervical cells deposited in the multi-cell device, whereas we only place some cells with cancer (that is, enough quantity to be visualized in a field for the purpose of the example), they are subsequently placed and It is grown for growth at 37 ° C. To activate and make reproducibility tests and have the samples by Duplicate, in triplicate or have samples in quadruplicate. The cells are treated inactivating with peroxidase before being labeled with antibodies. The Her2 monoclonal antibody is added as the main antibody in 1 μg / ml in PBS. It is incubated for one hour at 37 ° C. And for detection an anti-mouse IgG antibody (diluted at 1 μg / ml in PBS) is added and incubated another hour at 37 ° C, then ligated to the streptavidin which is a horseradish peroxidase (also diluted to 1 μg / ml in PBS is incubated for another hour at 37 ° C). The reaction develops with the addition of DAB substrate, producing a brown staining and is stopped with PBS containing azide. The multi-cell device is evaluated with the microscope at different magnifications (Figures 3A-3E: 10 X; Figures 3F-3 G: 40 X).

Figure 3A: Sample containing a mixture of normal human cervico-uterine cells and human cervical-uterine cells with cancer (indicated by a red arrow), which is processed with the methodology and the multi-cell device object of the present invention, stained with hematoxylin and labeled with anti-Her2 antibody (10X magnification) Figure 3B: Sample containing a mixture of normal human cervico-uterine cells and human cervical-uterine cells with cancer (indicated by a red arrow), which is processed with the methodology and the multi-cell device object of the present invention, stained with hematoxylin and labeled with anti-Her2 antibody (10X magnification) Figure 3C: Sample containing a mixture of normal human cervico-uterine cells and human cervical-uterine cells with cancer (indicated by a red arrow), which is processed with the methodology and the multi-cell device object of the present invention, stained with hematoxylin and labeled with anti-Her2 antibody (10X magnification) Figure 3D: Sample containing a mixture of normal human cervico-uterine cells and human cervical-uterine cells with cancer that is processed with the methodology and the multi-cell device object of the present invention, stained with hematoxylin and labeled with anti-cancer antibody. -Her2 (10X increase) Figure 3E: Sample containing a mixture of normal human cervico-uterine cells and human cervical-uterine cells with cancer (indicated by a red arrow), which is processed with the methodology and the multi-cell device object of the present invention, stained with hematoxylin and labeled with anti-Her2 antibody (10X magnification) Figure 3F: Sample containing a mixture of normal human cervical uterine cells and human cervical uterine cells with cancer (indicated by a red arrow), which is processed with the methodology and the multi-cell device object of the present invention, stained with hematoxylin and labeled with anti-Her2 antibody (40X magnification) Figure 3G: Sample containing a mixture of normal human cervico-uterine cells and human cervical-uterine cells with cancer (indicated by a red arrow), which is processed with the methodology and the multi-cell device object of the present invention, stained with hematoxylin and labeled with anti-Her2 antibody (40X magnification) The results show that the use of antibodies improves the recognition of cells with cancer, in comparison with the morphological evaluation alone.

Table I: Immunostaining of cancer cells between normal cervical cells in solution using the ELISA method which is the format commonly used in tests performed with antibodies, As in figure 3 (examples 2 and 3), breast cancer cells are mixed with Normal cervico-uterine cells of a patient from a ThinPrep sample, except that in this case a certain number of cancer cells are used and they are increasing using (200, 300, 400, 600, 800, 1200 cells per cell). In addition, this experiment is carried in the multi-cell device, which is a plate with 96 cells, in this format it facilitates that the tests can be alternated with immunostaining, being able to practice both methodologies, this is practiced with the cell in a suspension in water base, as described in detail in Example 4. Immunostaining is as described in Figure 3 with duplicate samples, except that the substrate used for detection is soluble, such as TMB and the antigen-antibody reaction it is evaluated by a colorimeter that is a 450 nm reading plate. Controls include a white suspension without cells and a white suspension with cells but without any primary antibody. OD 1 and 0 D 2 readings are performed on the wall of the cell where the sample is in suspension of whole cells (containing a cell pellet) luminous), or only in the supernatant of the cell, respectively.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the disclosure of a completely new methodology and device for the detection and management of cervical-uterine disease in cervico-uterine samples of patients.

Multi-cell device The "multi-cell" device of the present invention consists of a solid support with multiple separated circular areas, with a patient sample in each one, carrying out with this device and methodology a multiple housing for a simultaneous evaluation of multiple samples of Patients in screening for the detection of cervical-uterine disease, using the methodology of the present invention.

To the extent, the novelty of the device is that it allows the evaluation of multiple cervico-uterine samples simultaneously, in comparison and contrary to the conventional pap smear or current gold standard to prepare analyzes, whereby individual samples will be placed in a glass slide and stained using conventional Papanicolau stain.

Figure 1 illustrates the prototype of the multi-cell device of the present invention with numbering of the (1-40) circular areas (or cells) of 15 mm in diameter.

This invention covers other formats are encompassed, such as, but is not limited to: 6, 12, 24, 48, 96, 384 wells. It is known that workers skilled in the art have adjusted the diameter of the device and the format of the methodology (for example, the number of cells), to the scope of application of the test, are relevant changes based on the cell. Modifications of the multi-cell device were made to facilitate the reading in the microscope, the handling or manipulation of the sample for the test, as well as the modifications for that. The multi-cell device is compatible with any instrument or equipment that may be associated with the use and application of the device, whether for manipulation, qualitative or for quantitative measurement purposes.

The multicell device is preferably made of polycarbonate, however, the present invention can also be made in other materials, such as but not limited to: glass, acrylic, flexible glass, polystyrene, polypropylene, etc. The modifications in the material have what to do with the facilitation of protein adsorption, and to ensure compatibility with the organic or aqueous solutions of the process, and other modifications as is known to workers skilled in the art, are included in the present invention.

The multicell device is coated with a layer of thin paint, therefore, it creates and delimits each of the circular areas that make up the multicell device, so that no contamination can be produced or cross between the samples. As the layer covering the device can be of different thickness, the circular areas (multicells) can be delineated by a ring of different thickness and height, converting the area into a well of different depth.

The methodology of the present invention allows the simultaneous analysis of multiple samples.

To this extent, the multicell methodology and device object of the present invention has utility application as a new high performance format for a variety of other cell-base tests, including conventional cytology and cellular immunostaining, which are frequently performed in individual glass slides. The methodology and the multi-cell device object of the present invention can be used preferably in the cervico-uterine Pap test of the national screening programs.

For purposes of the present invention, the cervical cell sample is intended to be a sample collected directly from the cervix of a patient that includes cells, tissue or body fluid, using any device, for sample collection which includes but is not limited to a cytological brush, spatula, scraping or secretions from an area or through a needle to aspirate body fluids. Cervical specimens may also come from but not limited to fresh or frozen human tissues, paraffin-fixed tissue, a biopsy specimen, exfoliated cervico-uterine cells, or samples of cervical-uterine mucus.

Moreover for the purposes of the present invention, we understand that cervical samples include cervico-uterine cells, which come from samples taken and suspended in water base such as, but not limited to, ThinPrep. RTM. Preparation (CYTYC, Inc.), or SurePath. RTM.

(TriPath Imaging, Inc.).

Also included in the present invention are the suspension of cervical cells using other liquid-based cytology-conserving solutions, such as alcohol-based cytology or other solutions of other type for immunohistochemistry tests, or cervical cells in suspension in non-aqueous solutions. preservatives, such as phosphate buffered saline (PBS). Cervical cells collected by any technique mentioned or obtained from any of the solutions mentioned above and then placed in a slide cell, which includes a monolayer in each cell, as contemplated in the present invention.

In particular the inclusion present cervical cells included in the methodology of the invention come from aliquots of ThinPrep. As an alternative, the aliquots of ThinPrep are centrifuged, washed in PBS, re-segmented through a gentle centrifuge, finally in the next step they are resuspended in PBS.

Furthermore, any other biological fluid that contains cells and that can be evaluated by cytological staining, microscope examination, immunostaining with a specific antibody or any combination of antibodies are also included in the present invention. These biological fluids include but are not limited to: bronchial-alveolar lavage, sputum, gynecological smears, nipple aspirate fluids, urine, etc. In many cases, these biological fluids are associated with the detection of diseases and the conditions of specific organ sites, for example, bronchial lavage. alveolar for asthma or lung cancer and / or any lung disease, fluid sucked from the nipple for breast cancer, urine sediments after a digital rectal examination for prostate cancer examination, etc. Included in the present invention are the cells derived but not limited to, fresh or frozen human tissues, tissue fixed in paraffin, sample of the biopsy, micro-cuts of tissue tumor etc. Therefore, the methodology and the multicell device of the present invention have applications useful in the detection and possible detection of a variety of diseases, beyond gynecological and cervico-uterine diseases.

Automation, As those skilled in the art know, this device object of the present invention, can be used as a new high performance methodology can be used in a manual, semi-automated or fully automated format. The classic staining, which includes the staining of the Papanicolaou test used in the national screening program for the detection of cervical disease, as well as immunohistochemistry procedures that are applied to several lamellae, have already been automated for a long time. procedures that allow the handling of a large number of multiple samples per hour. Included in the present invention are systems, apparatuses, methods and modifications allowing automatic control thereof and scheduling of all tasks necessary to increase the performance of automated procedures in the multi-cell device, such as but not limited to a: shows the deposition, blowing air, washing, staining in various baths at specific intervals, Addition or elimination of reagent bottles or containers of liquids, mixture of reagents, application of reagents for the multi-cell device, introducing with this a new solid support (ie, the mutli-cell device). with additional samples, increasing performance, having a solid support throughout the procedures performed, capturing images of the device, measuring colorimetric reactions, etc.

Partial or total automation of the processes described in this document are carried out by the present invention.

Cytological stains using the multi-cell device object of the present invention.

First, the methods of the present invention comprise the use of the multi-cell device in conventional cytological staining. In fact, in an example that is preferred to use the present invention, the application of the multi-cell device in the classical hematoxylin stain is demonstrated (For example 1, Figures 2A-2D).

They are cervical cells that come from a normal patient sample in Thin Prep, deposited in one of the cells or circular area of the multi-cell device. Several samples can be stained simultaneously and visually evaluated under the microscope. Therefore, the multi-cell device has utility and application to convert a variety of other cell-base tests into a high-throughput format, including the detection of cervical-uterine disease in national screening programs.

In carrying out this practice, the multi-cell device has been irradiated with UV rays to enable data attached to the cell.

Methods for coupling the alternating cells, a method known to workers skilled in the art (such as, but not limited to, polylysine, Union agents, plastic treatment for cell adsorption, etc.) are carried out by the present invention, depending on the material from which the multi-cell device object of the present invention is made.

Workers skilled in the art know that the size of the circular areas of the multi-cell device must be reasonably compatible with the number of cells to be examined. Therefore, depending on the scope of the application of the experiment, between visualization, detection or national screening program, a sample with a determined number of cells and the volume that can be reasonably hosted and spread on the surface of the well will be chosen. cell of the selected multi-cell device. For example, a diameter of 15 mm may well accommodate about 300,000 cells which is the number that may be necessary for an accurate diagnosis in the Papanicolaou screening test.

After depositing the cells in the multi-cell device, the cells are dried with RT, or alternatively exposed in a chamber at a temperature of 37-40 ° C, then rinsed with PBS. The cells are cleaned of excesses or debris, with this procedure a noticeable deterioration of the smear will be observed to prepare the staining of the cervical sample. In a specific protocol, hematoxylin staining Example 1 is performed (FIGS. 2A-2D), thus coloring nuclei of cells in blue and visual evaluation is carried out under the optics of the microscope.

The results, as shown in Figures 2A-2D, demonstrated that cytological staining can be performed successfully in the high-throughput multi-cell device format of the present invention.

Application of the multi-cell device for the detection of cervical cancer.

The device of the present invention has utility and application of high performance in the detection and in the national detection programs in screening of the cervical disease.

Because preneoplastic, dysplastic and cancerous lesions are rare in human cervical samples, this particular protocol of the present invention resorts to an experimental condition imitating a sample of some patient for the detection of cervical cancer, mixing the normal cervical cells with the cells of a cancer cell line grow together overnight in a co-culture (example 2).

As described in detail in Example 2, we have cervical cells that come from a normal patient sample prepared with Thin Prep, are deposited in a single circular area (cell) of the multicell device and treated as in Example 1 (ie, air dried and rinsed with PBS). Then there are the cell layers coming from a line of cancer cells in each of the individual circular areas (cells) of the multicell device and they are incubated ON at 37 ° C to spread and adj.

The next day, the cells are fixed with 95% ethanol to ensure the attachment of cancer cells and stained with hematoxylin. Multiple cervical patient samples can be used simultaneously in the multiceld device, and the co-cultures can use different amounts of cancer cells with respect to normal cells.

In this specific protocol of the present invention, we put a line of breast cancer cells and BT474 is used, but any line of cancer cells can be used for this purpose. The co-cultures of some cancer cells between normal cervical cells redoing the test conditions of a patient sample for the screening of cervical cancer, where a few abnormal cells should be recognized in a field of normal cells.

Results of the microscope evaluation in the observation of round or elongated cancer cells (here the epithelial cells of a ductal carcinoma) that have grown between squamous cervical cells, characterized by its large polygonal shape and that based on the blue staining of the cells. nuclei with hematoxylin. Because in this example, cervical cells and cancer cells are both stained with Hematoxylin, they can only be distinguished based on cell morphology alone. The difference in cell shape between cancer cells and normal cervical cells can be seen in Figures 3A-3 G infra. In contrast, in the following protocol (namely, Example 3, Figures 3A-3G), cervical cells are stained with hematoxylin, while cancer cells are stained with a specific antibody. Therefore, cervical cells and cancer cells are distinguished based on the morphology of the cell and on the basis of specific immunostaining.

In this specific protocol, hematoxylin staining is performed. However, the present invention encompasses staining of normal and abnormal cells in the well device with any cytological staining, including the Papanicolaou stain used in cervical screening. To the extent, the device and method of the present invention finds utility application in the detection of cervical disease and in screening.

In conclusion, this specific protocol of the present invention confirms that co-culture and Hematoxylin staining of a few cancer cells between normal cervical cells can enable microscopic evaluation of cell morphology differences, serving as an experimental model of a patient sample for the detection of cervical cancer.

The co-culture model in this specific protocol of the present invention can be applied to the cells of other biological fluids, except the cervical smears and Thin Prep, such as but not limited to: bronchioalveolar lavage, sputum, fluid aspiration of the nipple, cerebrospinal fluid, peritoneal fluid, plasma, serum, semen, prosthetic fluid, etc. To the extent, the multicell device can be applied successfully to the examination of these biological fluids that may be associated with the detection of diseases and Different conditions in different organs, the extension of the useful application of the multiceld device to diseases and screening programs goes beyond gynecological diseases. As those mentioned above, other cancer cell lines and tumor cells can be used in conjunction with other biological fluids, whereby the present invention encompasses several other cancer models or screening and disease selection.

Immunostaining cervical samples using multiceld device and antibodies used in cervical screening, there is a need to improve the accuracy of the Pap test. Immunostaining with antibodies represents a more "objective test" than subjective visual reading of cell morphology.

In this document the term "antibodies" refers to a polyclonal recombinant, monoclonal antibody, full size molecule or an antibody fragment, including but not limited to FAB, scFv, single chain variable fragment, affibodies, diabodies and any other Antibody fragments that bind to the antigenic binding determinant site. The term "antibodies" is used interchangeably herein to refer to any of the species above. Therefore, antibodies include antibodies produced in vitro as well as antibodies generated in vivo in a mammalian species capable of immune response. Methods for monoclonal, polyclonal, recombinant antibodies and their fragments are known to those skilled in the art (Colligan et al, current immunology protocols, Wiley Intersciences; Kohler et al. 256: 495 of nature-497, 1975; Phage visualization of peptides and proteins: a laboratory manual, Kay b. b., j. j. & Winter McCafferty, Eds, Academic Press, 1996).

Therefore, immunostaining may serve as a complement to the morphological interpretation offered by the pathologist. Antibodies against specific biomarkers can help in the diagnostic interpretation of preneoplastic and dysplastic lesions in the detection of cervical cancer, increasing the accuracy of pathological anatomy or diagnosis based on cytology.

Therefore, in a preferred protocol of the present invention, it is the simultaneous immunostaining of multiple cervical samples that is performed in the multicell device of the present invention, using commercially available and used antibodies (example 3f Figures 3A-3G). Those who understand or are those skilled in the art, know that the target antigens of the antibodies used for the diagnosis, can be in different locations of the cell, ie, nucleus, cytoplasm or membrane, can also help the pathologist in the diagnosis that is based on cytology or histology alone.

This incorporation leads to the experimental conditions that were established in Example 2, which are relative to a mixture of normal cervical cells simulating a patient sample for the detection of cells with cervical cancer, since the preneoplastic, dysplastic and Cancerous tumors are rare in cervical samples.

The cell mixture is treated with 95% ethanol to ensure that the cell adheres to the surface of the multi-cell device during the procedure, however other protocols known in the state of the art are carried out by the present invention for guarantee the results.

In this specific protocol, the multicell device allows the simultaneous determination of multiple compounds by a constant number of normal cervical cells and an increasing number of samples of cancer cells. > In this example, the line of breast cancer cells that was used was the BT474, however, the present invention encompasses any preneoplastic cell or neoplastic line or tumor cells. Mixtures of cells, with varying rates of cancer cells and normal cells, are performed in triplicate to check reproducibility and treatment for immunostaining.

You can also prepare several samples from the same patient if studied with different antibodies and with different staining patterns (either nuclear, cytoplasmic or membrane) are used to confirm the diagnosis better.

In the preferred protocol of the present invention, the main antibody that is used is a monoclonal antibody against Her 2, which is a membrane glycoprotein with tyrosine kinase activity belonging to epithelial growth factor receptors known to be over-active. expressed in some patients with breast cancer. However, the method of the present invention encompasses the antibodies, defined above and others such as (for example, the culture of polyclonal cells, monoclonal, supernatant, purified or not, recombinant, etc.). And variant, against any antigen in any location of the cell.

In the examples detailed herein, the immunostaining procedure comprises the use of an immunoglobulin which is a bio-staining anti-mouse antibody that is used as a secondary antibody followed by streptavidin linked to a horseradish peroxidase, followed by last by the addition of a precipitating substrate the DAB. After showing the nuclei of the cells stained with hematoxylin which are observed blue in the multiceld device that is observed under the microscope.

Other methods of immunostaining, known in the field, can also be carried out in the multicell device object of the present invention.

For example, protocols that are based on different indications and different detection systems, such as alkaline phosphatase, biotin-streptovidin, or fluorochromes, can also be successfully performed within the scope of the application of the present invention. On the other hand, while cervical cells must undergo a pre-treatment inactivation by an endogenous peroxidase, if peroxidase-based staining is used, the aforementioned pre-treatment is not necessary when fluorescence-based imaging system is used , then the protocol is modified accordingly.

Alternative modifications known to those skilled in the art are carried out by the present invention, such as but not limited to the following: any method for making antigens that bind more easily to the antibodies can also be used in the practice of the invention , including antigen retrieval methods, known in the art, alternative substrate selection protocols and alternative staining methods to block endogenous or nonspecific biotin binding (see also).

According to the method of the present invention and as shown in Figures 3A-3G, the multi-cell device object of the present invention can be used to discriminate a few cancer cells between normal cells in a mixed population, in an experimental condition simulating a cervico-uterine sample for the detection of the Papanicolaou test. In fact breast cancer cells stained brown (DAB) by the anti-Her2, antibody in a field of squamous cervical cells, characterized by its polygonal shape and Hematoxylin had stained blue nuclei.

It is emphasized herein that, while the method of the present invention is exemplified that commonly uses antibodies that are specific to cancer cells, the use of other antibodies against biomarkers that are overexpressed or differentially expressed in cells with lesions dysplastic, preneoplastic or malignant cervical cells, such as antibodies against p6 (INK4a), Ki-67, HPV, any other cervical-uterine cervical-uterine infectious disease, or antibodies that react with cervical cells in the disease or condition normal, it is also encompassed in the present invention.

In conclusion, the method of the present invention depends on an antigen-antibody target interaction to identify malignant cells in the patient specimen. To the extent, the device and methods of the present invention have utility and application as to improve the potential of the Pap test, and as a new cervical-uterine screening of high-performance immunocytology.

The methodology of the Immunostaining of cervical-uterine tissue samples using the ELISA-type cell format. Another method of the present invention relates to an immunostaining method based on a sample of human cells and an antibody (or a combination thereof) in a multi-cell ELISA type format.

When the cell and the antibody to the combination thereof coming from a suspension is able to detect cervical dysplasia, neoplastic or preneoplastic lesions or their modifications in a sample of cervical tissue, the method of the present invention has utility and application in the detection of cervical cancer and national screening programs.

As explained above, depending on the cell suspension used and the antibody or a combination of antibodies is used, then, the method of the present invention has utility application in the detection and screening of other conditions and diseases, more beyond gynecological diseases, including cancer of other organs.

In a preferred protocol of the present invention, the immunostaining method of the cell of the present invention using an ELISA-type format is applied to detect cancer cells simultaneously in multiple samples of cervical tissue (example 4, table 1) ).

In the preference of this protocol, the concept of "multi-cell" device format, which has been extensively described so far in the present invention, is extended to the common type of ELISA (multi-cell) format, such as a 96-cell plate or its equivalent. Plates with a different number of cells or wells per plate can be imagined, including but not limited to 6, 12, 24, 48, 96, plates of 384 cells or wells, which allows it to be cost effective, easy to use and small scale to the high performance format, since considering this case all the procedures are included in the present invention.

In addition, this document emphasizes that while the 96 cell or well format could be the most common and easy-to-use high-performance format, other quantities and sizes may be more compatible with the number of cells required. necessary. for a cervical detection, about 300,000 cells must be evaluated to provide an accurate diagnosis. In fact, skilled workers in the art are known to well match the diameter of the assay format (e.g., the number of cells) to the scope of application of the corresponding cell-base assay.

While an important application of the present invention is high throughput screening, the present invention also encompasses the application of this immunostaining assay of a cell sample from a different version of suspension, as well as fluoximetry assays.

Note that, while the multicell device of the present invention described above involves a mixture of adherent cells, the ELISA-Multi-cell type format may involve a mixture of adherent cells, as well as a mixture of cells in suspension. Adherent cells made through different methods, including alcohol fixation or air-drying, can be housed in a variety of cell plates or wells, while cells in suspension can preferably be treated at the bottom of the cell. the plate, and they should be centrifuged at each step of the procedure.

Note that, as described below, when dealing with cells from a suspension, each step of the procedure is followed by a gentle centrifugation step to ensure that the cells adhere to the bottom of the cell or well , as detailed in the example.

Immunostaining follows the principles of Example 3 (Figures 3A-3G), except by using a specific number of cancer cells in the mixture, antigen-antibody reactivity can be quantified by colorimetric reading, rather than by a visual reading Semi-quantitative based on microscope examination.

The present invention encompasses any modification of an ELISA-Multicell type plate, related to the format, whether the number, the size and the support material of the plate. Moreover included in the present invention are support modifications deemed necessary to be compatible with or to facilitate reading in the microscope, for its handling and manipulation, the readers of immunoassay, handling, manipulation and any other instrument or equipment that may be associated with the use and application of the present invention either for handling, or for measurement purposes qualitatively or quantitatively for measurement purposes.

On the other hand, the present invention. covers any modification of the methodology and the device to be able to develop versions to perform manual procedures, semi-automated procedures and fully automated procedures. The automation procedures will vary depending on: The automation of the assay will depend on whether there are adhered or suspended cells in the assay, and all the modifications are included in the present invention.

In this protocol of the present invention, a constant amount of cervical cells was preferred and a cervical-uterine sample from a normal patient in (Thin Prep) is deposited in each cell of the multi-cell ELISA-type device, a format that involves planting a increasing number of breast cancer cells of BT474, in a co-culture, and performing an experiment simulating a patient sample for the detection of CACU., as described in example 2.

The mixtures are prepared in triplicate to allow quantification.

The cells are treated with H202 in to achieve inactivation of the peroxidase before staining the antibodies, if it is a method related to the use of peroxidase in the staining procedure. Then, immunostaining with specific antibodies is carried out.

The primary antibody used here for the purpose of this example is an anti-Her 2 (located on the membrane), followed by secondary antibodies conjugated with biotin and conjugated with streptavidine peroxidase. The antigen-antibody complexes are visualized by the addition of a soluble substrate, such as, but not limited to, tetra-methyl-benzidine (TMB). Immunoreactivity is directed by measuring the optical density (OD) of the colorimetric reaction through a plate reader at the corresponding wavelength.

Other immunodetection systems are carried out by the present invention. The results of this experiment are shown in Table 1 and show us the immunoreactivity, measured by the reading, which increases directly proportional to the number of cancer cells in the cell reviewed. The duplicate of the samples was indicated for the experiment to reproduce.

The method of the present invention offers some advantages over the ELISA test of conventional type. In fact, in the conventional ELISA procedure, the protein extracts of cells are fixed by a layer in the Multicells plate, instead of fixing the intact cells. Therefore, biomarkers must be soluble to be detected in an ELISA assay. On the contrary, whether it is soluble or not, it does not matter which biomarkers are detected in an intact cell. The advantage identified above is particularly important, as they have several publications that point to the poor quality of protein extracts derived from cervical cells (Ding, 2008; Ge Y et al., High-level proteomic analysis that identifies dysplastic cervical cells obtained from the lamellae of the ThinPrep and the use of laser for micro dissection and fix it with mass spectrometry, J Proteome Res 6: 4256-4268, 2007).

The poor yield of protein extracts obtained from cervico-uterine cells can affect the accuracy and reproducibility required for a conventional ELISA assay.

It is known in the art that currently visual detection in the cervico-uterine samples presents a significant variability in the observation between different observers. In a study carried out previously in the present invention, that is, the immunostaining of the cells in the multi-cell format device, we have shown that the use of antibodies provides greater precision on the evaluation of cell morphology on its own. In this project specifically, that is, the immunostaining of the cell using a device with ELISA Multi-cell format, we are more easily shown that the immunoreactivity can be quantified especially through a colorimetric reading and the test is perfectly reproducible .

In the context of conventional screening for cervical-uterine disease detection, where significant well-prepared human resources and equipment are required, for reading colorimetry to read immunostaining of cervico-uterine cells is a new application of utility of the present invention.

It facilitates the evaluation of the sample by facilitating the colorimetric reading, eventually offering an alternative to the pathologist's evaluation.

In conclusion, they show us that the applications of the utility of the present invention are: SIMULTANEOUS MANAGEMENT OF VARIOUS SAMPLES IMMUNOTINTION THAT REPRESENTS AN IMPROVEMENT ON THE SCREENING PERFORMED WITH THE CONVENTIONAL PAPANICOLAOU FOR THE DETECTION OF CERVIC-UTERINE DISEASES, AS WELL AS THE CURRENT PRESENTATION OF THE SAMPLE ThinPrep In addition kits are provided for practicing the methods of the invention.

EXAMPLES The following abbreviations are used throughout. ddH20: double bidistilled water HR: hour; min: minutes; sec: seconds; rpm: rotation per minute; RT: temperature.

Example 1: Staining of the cervico-uterine samples with Hematoxylin with the multi-cell device, a fraction of a single sample of thin PReP (ie250 μ1 volume, approximately 50-100,000 cells) is placed in layers in a single circular area (cell) of the multi-cell device. Therefore multiple samples can be evaluated simultaneously. The device has been previously irradiated with UV rays, which allows us to place the sample of the cells. However, the methods are alternated to be able to place more samples of cells depending on the material of which the device is formed.

The samples are dried with RT air or in a chamber at temperatures of 37-40 C, then the samples are rinsed with PBS. Washing and removing excess debris from the cells, leaving the cells ready for any cytoplasic stain.

In this specific assay, hematoxylin staining is performed. For staining with hematoxylin in the multi-well cell device the following steps are followed: 1. - Rinse with ddH20. 2. - Cover with a few drops of weak Mayer base hematoxylin solution for multicell device of 1-2 minutes, rinse twice in the tap with H20 or buffer solution (tap water, or with sodium or lithium carbonate solution to ensure alkaline pH) and staining the nucleus of the blue cell and rinsing once with ddH20. And the multicelled device is observed under the microscope using different amplifications (Figures 2A-2D).

Example 2: Co-culture and hematoxylin staining of cervical cell samples from patients (from a sample from Thin Prep) and cancer cells in the cells of the multi-cell device, in an assay that mimics detection in Papanicolaou screening First, a volume of 250 μ? of an individual sample of Thin Prep, previously diagnosed as normal, in layers in a single circular area (cell) of the multicell device. The samples are air dried and then rinsed with PBS and stained with hematoxylin, as described in example 1. The breast cancer cell line BT474 (ATCC # HTB-20), similar epithelial cells, ie , of a breast ductal carcinoma, and is used in the co-culture experiment that would also include cervico-uterine cells from samples of patients from a Thin Prep. The cell lines were cultured in ATCC medium according to a recommendation of the supplier, trypsinized and with a standard procedure arrangement.

The BT474 cells are deposited in each individual circular area (cell) of the multi-cell device and several hours on at 37 ° C to scatter and place them in an oven. The next day, the cancer cells are fixed with 95% alcohol and stained with hematoxylin, as described above.

Results of the evaluation in the observation under the microscope of the cancer cells are observed elongated, which have grown between large polygonal cells squamous cervico-uterine cells, which are visualized by their nuclei stained blue. In this example, cancer and normal cervico-uterine cells are distinguished based on cell morphology alone. These results confirm that the co-culture of normal cervico-uterine cells and cancer cells can be used as a model to perform the Papanicolaou test.

Example 3: Immunostaining of the cervico-uterine sample of patients performed in the multi-cell device using monoclonal antibodies.

This example describes how immunostaining can be performed in the multi-cell device using commercially available monoclonal antibodies. In this example, the usefulness of immunostaining as a complement to the microscopic evaluation of the cell morphology in screening for cervical-uterine disease is demonstrated.

Given that preneoplastic, dysplastic and cancerous lesions are rare in tissue samples from Thin Prep, we have resorted to an experimental condition imitating a patient sample for the detection of cervical cancer, ie. Place a mixture of the normal cervico-uterine cells with the cells of a cancer cell line that are cultured ON in co-culture, as described in example 2. In this experimental model, a constant amount of cervical-uterine cells normal patient from a preparation of thin, diagnosed previously diagnosed (250 μ 1 volume) is placed in layers in each circular area (cell) of the multi-cell device, dried with air in RT or in a chamber at temperatures of 37- 40 C then, rinse with PBS. Subsequently, some cancer cells are added (ie, sufficient to be visualized in a field in order to perform the example) are layered, in triplicate and grown for growth at 37 0 C. The next day, the cells they are fixed with 95% ethanol, however, instead of proceeding with hematoxylin staining as in example 2 which is the previous example, the multi-cell device is prepared for the immunostaining procedure. In this example, breast cancer cells of the BT474 cell line were used and were labeled with monoclonal antibodies against Her 2 (using an adequate volume of 1 g / ml of the PBE solution, sufficient to cover the cells). Note that the cells must be treated with H202, until inactivating the endogenous peroxidase, as described in detail in Example 4 below, if a base peroxidase is used in the detection system. The primary antibody is incubated for 1 hour at 37 ° C. Next, for detection, a biotinylated anti-mouse IgG antibody is used as a secondary antibody (Jackson laboratory) diluted at 1 g / ml in PBE is added to each area of the multi-cell device and incubated for 1 hour at 37 ° C. The secondary antibody is followed by streptavidin linked to a horseradish peroxidase (Jackson Lab) diluted to 1 μg / ml in PBE without azide and again incubated for 1 hour. hour at 37 ° C.

The reaction is developed by the incorporation of the freshly prepared substrate of tetrahydrochlorite 3-3 'diaminobenzidine (DAB at 5 mg / ml). followed by incubation in RT. Developing a color that is checked under the microscope and the reaction was stopped with PBS containing 0.05% azide. Hematoxylin staining is counted by counting the nuclei of cells stained with blue, and the multi-cell device is observed under the microscope.

The controls necessary to perform this experiment include: a) Only normal cervico-uterine cells. b) Only breast cancer cells c) a mixture of normal cervico-uterine cells and breast cancer cells without immunostaining procedure. d) cancer cells alone marked or stained with anti-Her2 antibodies.

Figure 3A-3 G These figures show us how immunostaining allows a specific identification of the few cancer cells that exist in a sample that has normal cervico-uterine cells in the background, in an experimental protocol simulating a sample to detect cervical-uterine disease in screening through the Papanicolaou Test, in addition to the observation of cell morphology differences alone.

Example : Immuno-labeling of the cervical-uterine sample of the patient in (Thin Prep) and cancer cells in the solution, using the ELISA-type format and the commonly used monoclonal antibodies (figure 5).

In this example, the concept of the multicell device format extends to commonly known ELISA-type formats, such as a 96-well flat bottom plate or wells or equivalent. In fact, presenting different plates with different amounts of cells or wells can be provided, as it deems appropriate, that is, 6, 12, 24, 48, 96, or plates of 384 cells or pools, allowing in the small sample scale its high performance, is a cost-effective format easy to use, and accommodates the needs'. In addition, it is highlighted in this document that, although it could be the format of 96 cells or wells, it is the most common and easy-to-use high-performance format. Other quantities and sizes of cells or wells may be more compatible with the required number of cells needed for a cervical-uterine disease detection, such as up to 300. 000 cells that have to be evaluated to provide an accurate diagnosis.

On the other hand, while the assay device consists of a mixture of cells that adhere well, the 96-cell format well plate may include a mixture of adherent cells as well as a mixture of cells in suspension. -Keep in mind that when working with cells in suspension, each step of the procedure is followed by a gentle centrifugation step to ensure that the cells are collected at the bottom of the cell or well.

Finally, in this example immunostaining follows the principles of Example 3 (Figures 3A-3G), except that a specific number of cancer cells can be used in the mixture, the reactivity of the antigen-antibody complex can be quantified by means of of colorimetric reading, instead of a semi-quantitative visual reading based on microscopic examination.

In this experiment, a mixture of normal cervico-uterine cells and breast cancer cells is used, as described above in examples 2 and 3, (Figures 3A-3G), in this experimental protocol we simulated a sample of cells cervico-uterine for the detection of cervical cancer. And the procedure is as follows: First, we put a constant amount of cervical-uterine cells from a normal patient that come from a pre-diagnosed normal thin preparation (250 μl volume, approximately 50-100,000 cells) is deposited in each cell or well of a plate of ulti-cells, that is, a plate with 96 cells or wells for the sake of this example.

The cells that come from the preparation (Thin-Prep), should be treated to inactivate endogenous peroxidase. Plates containing samples of cervico-uterine cells are treated with an adequate volume of 1% hydrogen peroxide solution diluted in PBS (H 2 0 2, 100 to 300 SI / well according to the size of the ring, cell, well) for 30 minutes on RT with gentle Soft agitation 400 rpm. After inactivation of the endogenous peroxidase, the cells are rinsed three times with PBS containing 0.3 Tween 20 (PBST), centrifuged and the supernatant removed.

At this point, the cells are dried with dry air at RT and then rinsed again with PBS as described above in Examples 1-3, subsequently treated as adherent cells. On the other hand, they are gently centrifuged between each step, if treatment is given as if they were cells in suspension. An increasing number of breast cancer cells are placed from the BT474 cell line, the cancer cells (cancer from 200 to 1200 cells / well) is layered, duplicated and grown at 37 ° C.

The next day, the cells are fixed in alcohol with 95% ethanol, if the cervico-uterine cell samples have previously been dried with dry air, or if they had previously been centrifuged. In this example it can also be successfully performed, by layers with the same volume of cervical cells and with a determined number of cells with cancer, followed by a treatment of inactivation of endogenous peroxidase. Then, the cells are dried with dry air and washed with PBS, or simply centrifuged, depending on the procedure (either the fixed cells or the cells that come from a preparation in suspension) depends on which has been chosen.

To block the endogenous biotin of the cells, these are incubated for 30 minutes in RT with a solution of streptavidin in PBE without azide (10 SG / ml), followed by a rinse of PBST. The cells are then incubated her with a solution of biotin (200 SG / ml) in PBE (PBS with 1% BSA, 1 mM EDTA, 1.5 mM NaN3, pH7.4) and dried. The cells are rinsed three times with PBST, centrifuged and the supernatant is removed.

After mixing the cell layers and carrying out the inactivation of the endogenous peroxidase, the biotin is also blocked, finally immunostaining is carried out on the plate.

All the incubation steps have a duration of 30 min and are performed in RT at less than 400 rpm, and thus are shaken by the rest of the procedure.

The plates are incubated in a relevant manner with anti-Her-2 primary antibodies (50 Sl / ring, cell or well and a plate with 96 rings, cells, or wells, or those that are necessary to include all the cells). ), add a solution with an appropriate dilution in the PBE buffer for 30 minutes, in RT. The cells are washed three times with PBST to remove the primary antibodies that were not fixed (specifically, the rings, cells or wells are filled with buffer, and the plates are centrifuged for 10 minutes at 1500 rpm and the supernatant is separated).

A secondary antibody is conjugated with biotin (for the purpose of the invention of a biotin conjugated with an anti-mouse IgG antibody), then added to each ring, cell or well (50 SI of a lSg / ml solution of the secondary antibody biotinylated in buffer PBE) and the cells are incubated as previously described (for 30 min, in RT, at 400 rpm). The cells are washed three times with PSBT to remove excess secondary antibody as described above. The cells are turned down for 10 minutes at 1500 rpm, and the supernatant is carefully aspirated, preferably under vacuum.

A peroxidase conjugate with streptavidin is then added to each ring, cell or well (50 SI an lSg / ml dilution in PGBE buffer without azide) and the cells are incubated 30 min at RT and shaken at 400 rpm, as described above. . After washing with PSBT and two washes with PBS, the antigen-antibody complexes are visualized by adding a solution of 50S1 of TMB substrate to each of the rings, cells or wells, then incubation of 10-40 min in RT and shake at 400 rpm. Within the next 10 minutes a blue color will appear with varying intensity that will depend on the number of cells per ring, cell or well and the concentration of the antigen. The reaction is stopped by filling each ring, cell or well with H2S04, passing the cell suspension to a yellow color, the immunoreactivity of the plate being read in a colorimetric plate reader at 450 nm.

The examples presented above are merely intended to illustrate the various utilities and applications of this invention. It is therefore understood that numerous modifications and variations of the present invention are possible in the light of the foregoing teachings and, therefore, within the scope of the claims annex, the present invention can be practiced against what is particularly is publishing All patents and publications that are incorporated in this document for reference to the same extent as if each individual publication was specifically and individually indicating to be incorporated by reference. It should be understood that, although the present invention has been specifically published or disclosed by specific and chosen examples including optional features.

Modifications and variations of the concepts disclosed herein will have to be resorted to by those skilled in the art since such modifications and variations should be considered within the scope of this invention.

Claims (13)

1. A method and device to facilitate the simultaneous analysis of several samples of human biological material for the detection, management or for the national programs of detection of the disease, which are contained in each ring, cell or well, through which the cells are analyzed. , of the samples that can be fixed or treated in solution, through the single cytological staining or by staining 'by means of immunohistochemistry, using an antibody or a combination of them, through the quantitative or semiquantitative measurement of a visual evaluation, and where said methodology and device can be practiced manually, semiautomatically or completely automatically.

2. A device to facilitate the simultaneous analysis of several rings, cells or pocitos containing samples of a human biological material for detection, management or national detection programs.

3. The device according to claim 2, characterized in that the samples of human biological material in which the cells are fixed in a solid preparation.

4. The device according to claim 3, characterized in that the cells are stained with cytological staining.

5. The device according to claim 4, characterized in that the biological specimens comprise cervico-uterine cells treated with Papanicolau stain for the detection of cervical cancer.

6. The device according to claim 2, characterized in that the modifications to automate the methodology and procedures using said device.

7. An immunohistochemistry method that contains a cell of a human being that comes from a sample containing biological material that is analyzed with an antibody or combination of antibodies and the device according to claim 3.

8. An immunohistochemistry method that contains a cell that comes from a biological sample is analyzed with an antibody or combination of antibodies and the antigen-antibody reaction is measured in solution.

9. The method of claim 7 wherein the biological specimen is a cervico-uterine sample.

10. The method of claim 8 wherein the biological specimen is a cervico-uterine specimen.

11. The method of claim 9, wherein the method is carried out as a complement to the conventional national pap smear screening programs, ie cytological staining for the detection of cervical cancer.

12. The method of claim 10 The method of claim wherein the method is carried out as a complement of conventional Papanicolaou cytological staining for the detection of cervical cancer.

13. A ki. to practice the methods of claims 12.