MX2013013365A - Integrated system of high performance for detecting gynecological diseases. - Google Patents

Abstract

The present invention describes a new device and in vitro methods for applying the same in a high performance detection process, detection and treatment of gynecological and cervical diseases. The multi-well device of the present invention consists in a solid support with multiple well-defined areas, each area housing a sample of a patient, which enables the simultaneous evaluation of samples. The methods of the present invention comprise conventional cytological staining, Pap stain of the cervix, and immune-chemical staining using antibodies or a combination of antibodies able to be bonded to the biomarkers overexpressed in cancer, including cervix carcinoma and dysplasia, compared to normal controls. The device and methods of the present invention may be practiced in any of the manual or automated modes, also being applied to any biological fluid or cell suspension of any biological sample as there is a wide variety of essays of cellular biology, and detection of diseases of the cer vix and further diseases. The present invention describes a new device and in vitro methods for applying the same in a high performance detection process, detection and treatment of gynecological and cervical diseases. The multi-well device of the present invention consists in a solid support with multiple well-defined areas, each area housing a sample of a patient, which enables the simultaneous evaluation of samples. Also includes the methods of the present invention.

Description

INTEGRATED SYSTEM OF HIGH PERFORMANCE OF DETECTION OF GYNOCOLOGICAL DISEASES BACKGROUND OF THE INVENTION Gynecological disorders are frequent and without medical attention in many communities that lack the resources and infrastructure to perform a sophisticated screening and diagnosis to the majority of the population. Especially in rural areas and some countries, the rates of women's disorders without medical attention represent the majority of the female population. On the other hand, cultural and economic factors impede access to preventive care.

Gynecological disorders encompass a wide range of serious pathologies, including autoimmune diseases, neurodegenerative conditions, viral and bacterial infections, and proliferations of precancerous and cancerous cells and tumors. Of this group, cervical cancer is the most serious.

Cervical cancer is of particular interest along with precursor conditions and associated risk factors such as HPV and other infections. Cervical cancer is the most commonly diagnosed cancer and the third leading cause of cancer death in women worldwide. There were 555,100 new cases and 309,800 deaths estimated in 2007, 83% of which occurred in the developing world (American Cancer Society, ACS Global Cancer Facts and Figures, 2007).

Since 1957, testing of samples of patients with Papanicolau stain, the so-called "Pap test" has become the routine test in the US. and in some developed countries. As a result, the incidence rates of cervical cancer has declined in all racial groups and precancerous lesions of the cervix are frequently detected leading to timely treatment and a significant decrease in mortality rates.

Infection with human papilloma virus (HPV) is a major etiological factor in cervical cancer. The magnitude of the risk association is greater than that of smoking and cancer of p > ulmon (Unger, ER, Barr E, human papillomavirus and cervical cancer, in Emerg Infect Dis 10: 2031-2032, 2004). Among the 200 known types of HPV, the HPV16 and HPV18 types are most commonly associated with cervical cancer, producing greater than a 200-fold increased risk (Castellsagué X, et al, worldwide virus etiology). human papilloma of cervical adenocarcinoma and its cofactors: Implications for screening and prevention, J Nati Cancer Inst. 5: 303-315, 2006). The causal relationship between HPV and cancer Cervical cancer has been exploited for the development of molecular technologies for viral detection to overcome the limitations of cervical cytological detection. HPV testing using DNA amplification is now used to complement misleading cytology in high-risk patients (Boulet, 2008). Currently HPV testing serves as a surrogate endpoint for the detection of cervical cancer. However, it has also been suggested for primary screening, especially in women over 30 years of age, followed by cytology if positive HPV (RA Smith, Cokkinides V, Eyre HJ, Guidelines of the American Cancer Society for the early detection of cancer, CA Cancer J Clin 55: 31-44, 2005). However, most HPV infections disappear spontaneously and only a small percentage of progression to CIN (cervical intraepithelial neoplasia or CIS (carcinoma in situ).) Other risk factors that contribute to cervical cancer can be immunosuppression, parity. high, smoking and nutritional factors, as well as the long-term use of oral contraceptives (/ ICO WHO Information Center on HPV and cervical cancer, Report on HPV and cervical cancer statistics in Brazil, 2007, ACS, 2010) The Bethesda system classifies cervical lesions precancerous as: 1) atypical squamous cells of undetermined significance (ASCUS), 2) low-grade squamous intraepithelial lesions (L-SIL) or cervical intraepithelial neoplasia (CIN I), characterized by mild dysplasia, 3) high squamous grade intraepithelial lesions (HSIL); and 4) cervical intraepithelial neoplasia, including carcinoma in situ (CIN II, CIN III / CIS) that is characterized by moderate to severe dysplasia. Note that LSIL / HSIL nomenclature refers to cervical lesions detected by cytology, while the nomenclature refers to the dysplasia of CIN determined in the histological analysis of biopsied cervical tissues (WHO / ICO, 2007).

CIN I rare (1%) becomes cancer, and most patients return to normal, even if left untreated, CIN II carries a risk of progression in cancer of 16% in two years and 25% after five years, if it is not treated. CIS is a confirmed neoplastic disease of the cervix that becomes invasive cervical cancer (CCI) for a period of 10 to 12 years. One year and five years of relative survival for patients with cervical cancer in the US it is 88% and 72% respectively, while the 5-year survival rate for patients diagnosed with localized cervical cancer is 92% (ACS, 2010).

The Papanicolaou test is a cytological staining of the cervical cells collected through a simple procedure performed in the doctor's office. Cells are stained directly on a slide, then fixed and stained (conventional Pap test), or first washed in a liquid preservative solution that thin the mucosa and remove cellular debris, before preparing a lamella (liquid-based cytology) , LBC). In both cases, the lamellae are read by a cytopathologist.

Papanicolaou test and LBC methods, which is based on the microscopic examination of individual cells in patient samples, have a limited sensitivity of 50% and high susceptibility to variation based on the subjective view of the technicians (Boulet GAV, et al., Human papillomavirus in the screening of cervical cancer: important role as a biomarker, Cancer Epidemiol Biomarkers Prev 17: 810-817, 2008). In a typical example, a technician can see 50,000 to 300,000 cells per slide to try to find 20-30 potentially abnormal cells, and double reading is often performed, which increases the variability. In addition, these tests require highly trained personnel and Well-capitalized laboratories, making labor-intensive and costly tests. The results of relatively low sensitivity to a high false negative rate, mainly due to inadequate sampling and preparation of inconsistent and inadequate lamellae. The development of the LBC technology (ThinPrep, Cytyc Inc., SurePath, TriPath Imaging, Inc.) has provided more standardized sampling method. For example, the processor Automated ThinPrep purifies the contamination of samples of blood cells, bacteria, mucus, and other inflammatory material, before depositing on a slide that will be analyzed by a cytotechnologist. This method detects 65% more LSIL in the general population of Conventional pap smear, and reduce by > 50% the number of inadequate cell samples, and provides the ability to perform additional tests outside the same vial. On the other hand, automated imaging systems currently identify suspicious cells that are subsequently examined by a pathologist.

Automated preparation of lamellae, automated reading methods, and the development of LBC technology have reduced the burden of analysis, as well as the intra- and inter-individual variability of evaluation. However, these technologies contribute to increase the high-tech infrastructure and the costs necessary for cervical screening, which clearly is not in the best interest of patients and health care providers in less developed geographical areas and low infrastructure settings. In these areas, individual visual readings of pap smears are still the norm.

According to the recommendations of the ACS (Smith, 2005), cervical cancer screening should be done every year with the Pap test or LBC. If the Pap test is abnormal and reveals ASCUS, the HPV test is performed, and if positive, the women refers to the colposcopy. If the Pap test reveals LSIL or HSIL, the women are immediately referred for colposcopy. Colposcopy is the microscopic examination of the cervix in acetic acid or Lugol stain to reveal abnormal cells, which can themselves be biopsied. Women over 30 who have had three consecutive tests with normal results followed undergo screening every 2-3 years with cervical cytology alone, or every 3 years with an HPV DNA test in addition to cervical cytology. Women older than 70 years and older who have had three or more normal Pap tests in a row, and there is no abnormal Pap test in the past 10 years, they can stop screening (Smith, 2005).

Tissue and serum biomarkers can improve the accuracy of Papanicolaou and complement current cervical research, and potential biomarkers have been described for cervical preneoplastic lesions and cervical cancer. Antigen Ki-67 is a nuclear protein at large expressed in proliferating cells (Goodson WH, et al The functional relationship between in vivo bromine-deoxyuridine labeling index and Ki-67 proliferation index in human breast cancer, Cancer Res. Treat chest May 1998 ... 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 all active phases of the cell cycle (late G1 -, - S, G2 and M), but absent in resting cells (G0 -). In diagnostic histopathology, antibodies to Ki-67 are used for the proliferation rates of grade tumors (Cattoretti G., et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIBland MIB3) detect proliferating cells. bh the microwave - processed formalin-fixed paraffin sections J Pathol 1992; .168: 357-63 ). Ki -67 immunostaining with the commercially available antibody MIB -1 has been evaluated as a test To increase the diagnostic accuracy of squamous intraepithelial cervical lesions (LSIL and HSIL, Pirog et al) Diagnostic accuracy of low-grade squamous intraepithelial lesions of the cervix has been improved with MILB - 1 immunostaining, Am J Surg Pathol 26:70 -75, 2002). However, as previously, the use of this marker is not accurate due to the interobserver variation in the diagnosis of LSIL.

The maintenance minichromosome (MCM) proteins, and particularly MCM-2, MCM-5 and MCM-7, are also useful for the detection of cervical disease including dysplasia and cancer (Williams et al., Proc Nati Acad Sci EE US 95: 14932-14937, 1998; Clin Cancer Res. 5: 2121-2132, 1999) Freeman et al., As demonstrated in conventional cervical smears or by immunohistochemical staining of cervical tissues. Recent results using a model of transgenic mouse HPV-have shown that MCM-7 seems more to be a specific marker for the detection of high-grade cervical disease by immunochemistry (Cancer Res. Brake et al, 63: 8173-8180, 2003 ..; Malinowski et al, Acta Cytol 43: 696, 2004; ... U.S. Patent 7,632,498 Malinowski et al, 2009) ..

Inhibitor-dependent kinase 2A (CDKN2A), also known as pl6 (INK4a) is a cycle regulator Cells overexpressed in preneoplastic lesions of the cervix that harbor HPV16 / 18 and in cervical cancer. p6 (INK4a) overexpression is due to the functional inactivation of the Rb retinoblastoma protein by the HPV E7 protein. p6 (INK4a) overexpression is therefore related to the expression of active HPV genes, instead of viral presence only. Thus, it has been proposed that overexpression of p6 (INK4a) can be used as a marker for persistent high-risk HPV infection and the detection of high-grade squamous epithelial lesions (HSIL; Klaes et al. (INK4a) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri, Int J Cancer 92: 276-284, 2001; Agoff et al, pl6 (INK4a) expression correlates with the degree of cervical neoplasia, a comparison with Ki-67 expression and detection of high-risk types of HPV, Mod Pathol 16: 665-673, 2003; Von Knebel Doeberitz et al, 2004, U.S. Pat. 6,709,832).

Immunohistochemical detection of p6 (INK4a) in cervical biopsies is routinely used to discriminate between HPV and associated non-HPV lesions (Redman R, et al., The utility of p6 (Ink4a) to discriminate between cervical intraepithelial neoplasia and non-p neoplastic equivocal lesions of the cervix, Arch Pathol Lab Med 132: 795-799, 2008), and as an indirect marker of high-risk HPV infection (Mulvany NJ, et al, the diagnostic utility of the pl6INK4a: a reevaluation of its use in cervical biopsies. : 335-344, 2008). In addition, pI6INK4a immunostaining of ThinPrep cervical specimens is being evaluated for its ability to aid in the identification of high-grade intraepithelial lesions, as assessed by follow-up biopsies and high-risk HPV DNA tests (Mcyer et al., The evaluation of the pl6INK4a expression in ThinPrep cervical samples with the pl6INK4a assay from CINtec, Cancer, 111: 83-92, 2007, Doeberitz et al, 2007, US Patent 7,306,926). Finally, a procedure based on ELISA for the detection of pl6 (INK4a) using protein lysates of the neck cells has been carried out. Exfoliative uterus to produce greater sensitivity and specificity for dysplasia and cancer compared to the ThinPrep test (Ding L, et al., ELISA test to detect CDK2A (expression of pl6 (INK4a) in exfoliative cells: a new tool for detection for cervical cancer, Mol Diagn Ther 12: 395-400, 2008).

Among circulating biomarkers, carcinoma of Squamous cell antigens (SCC) in serum correlates with tumor stage, tumor size, residual tumor after treatment, recurrent or progressive disease, and survival in cervical squamous carcinoma (Gaarenstroom KN, Bonfrer JMG. NACB, National Academy of Clinical Biochemistry: Guidelines for the use of oral markers in cervical cancer, 2007) . SCC antigen is a group of glycoproteins with a molecular weight of ~ 45 kDa, which belongs to the family of serine protease inhibitors. Two genes, SSC1 and SSC2, both located on chromosome 18q21.3, code for the neutral and acid isoform respectively. The neutral form is detected in both normal and malignant epithelial cells, while the acid isoform is found in tumor cells and in the serum of cancer patients with well-differentiated squamous cell carcinoma (Kato H, et al., The distribution heterogeneous of TA-4 acid in squamous cell carcinoma of the cervix, immunohistochemical demonstration with monoclonal antibodies, JPN J Cancer Res 78: 1246-1250, 1987). SSC1 and SSC2 are almost identical, they only differ in their reagent loops. There is evidence that regulate proteolytic events in both normal and pathological processes, however, have different biological functions (De Bruijn HWA, et al, El Clinical value of squamous cell carcinoma antigen in cervical cancer, tumor Biol.19: .505-516, 1998). Elevated levels of SCC have also been found in patients with squamous cell carcinoma of 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 the recommendations of the NACB (Gaarenstroom, 2007), SCC is not a sufficiently sensitive biomarker to detect cervical cancer, and its clinical utility in the prognosis and surveillance of response to treatment needs to be evaluated.

In conclusion, there is a need to improve the accuracy, performance and cost of current detection systems for gynecological disorders. The compositions, systems and devices of the present invention allow precision, low cost, high performance, and individualized rapid detection of gynecological disorders, with special application to cervical cancer. Because immunostaining antibodies represent an "objective test", antibodies against specific biomarkers for cervical cancer could thus aid in the diagnostic interpretation of LSIL by increasing the accuracy of the diagnosis of cytology or histopathology.

SUMMARY OF THE INVENTION The present invention describes new devices and systems for the detection of high performance and the detection of different pathologies, gynecological disease specifically in patient samples. Specifically, the invention provides a system for the extraction, verification, and high-throughput screening of samples to detect pathologies, including inflammatory cells for autoimmune disease, neuronal cells for neurodegenerative diseases and cells of the cervix. The present invention discloses an integral system for the collection of patient samples, coding samples for analysis and most of the results, analysis with proprietary biological markers, specifically using a high throughput screening process of centralized screening , and the notification of the results coded with the source of the patient's sample.

The high-throughput screening process includes the parallel processing of multiple patient samples using a common set of diagnostic techniques, including a unique set of markers for disease detection. To enable high performance detection, the invention includes a device " multi-well "comprising a solid support with multiple separate circular areas, each accommodating a patient sample, leading to the simultaneous evaluation of multiple patient samples with a common set of biochemical markers for high-throughput screening and screening The present invention includes a robotic manipulation device that transfers patient samples from conical tubes coded for the multi-well device to ensure standardization of the process and to allow high-throughput screening and disease detection. in the collected samples The present invention includes the results of entering diagnostic information in an internal database and delivery through the Internet portal to physicians, health providers and hospitals of the patient source where samples were collected clinics A new monoclonal antibody (mAb) capable of binding to a marker polypeptide for cervical cancer is disclosed, along with a defined epitope in which binding to the marker takes place. A list of present sequences is transmitted containing the amino acid sequence of the marker (SEQ ID NO: 1), the polynucleotide sequence of the gene for the marker (SEQ ID NO: 2) and the amino acid sequence of the epitope in the that the mAb une (SEQ ID NO: 3). The antibody may also contain labels and other functional entities that allow the detection or localization of the label, or the mAb, as well as for the detection of antibody binding to the label to form a complex in the epitope. Detection of the marker or antibody is carried out in the high-throughput format system for the processing of large numbers of samples and is carried out in parallel with other pap smears or cytological tests. Therefore, the application of the system in the detection, diagnosis and management of high-throughput disease of diseased cells integration of information processing and management, sample handling and processing, high-throughput processing biochemistry and microscopic analysis.

The high performance screening system includes the collection and processing of specimens derived from any biological fluid or tissue that is processed and screened in high performance format with the test protocols as described in this document for analysis, detection, diagnosis, management of the disease and detection of a variety of gynecological diseases and conditions.

The tamisa je of high performance of the device and systems of the present invention may also find utility application in a variety of other cell-based assays and cytology, as well as cell biology assays, including but not limited to the selection of drugs and small molecule compounds, and the study of cellular pathways The integrated high-throughput screening system is also composed of a new immunostaining format and patient sample processing equipment using the multi-well device assisted with the handling of robotic machines for liquid transfer and barcode coding that permeate the monitoring and integration of the information generated during this screening process in the database and information systems for health professionals. The patented combination of markers quickly and accurately distinguishes cervical cancer cells from normal cervical cells in a patient sample. On the other hand, a specific antibody also detects an antigen that is over expressed in cervical cancer cells. Therefore, the present invention takes advantage of the use of these specific antibodies in a high performance amisation format to identify cancer cells, which increases the accuracy of diagnosis of Significant way comparatively to the visual evaluation of cell morphology by the pathologist.

Also, encompassed by the present invention is the use of other antibodies or combination of antibodies against the biomarkers or combination of biomarkers that are associated with preneoplastic, neoplastic or dysplastic cervical cells. The use of antibodies against these biomarkers could greatly improve the current sensitivity of the Papanicolaou test.

Finally, the present invention includes a composition and a device for the detection of cervical disease and selection based on a cell suspension cell of patient samples. In the selection process, a plurality of conserved patient samples are analyzed by parallel processing in a high-throughput format followed by an analysis of the staining achieved with an anti-p6 (INK4a) antibody, which is located in the nucleus. of the cell and the cytoplasm. The tamisa e of high-performance robotic assisted that transfered the clinical samples of patients in a specific and precise manner and where the use of monoclonal and / or polyclonal antibodies against the specific biomarkers expressed by cervical cancer cells is contemplated. The present invention contemplates the use of a triple antibody combination against p6 (INK4a) , a biomarker indicative of the presence of cervical infection by the human papillomavirus, and anti Ki - 67, a biomarker known to be expressed in the nucleus of cancer proliferating cells, to facilitate the high - throughput diagnostic examination of patient samples In a preferred embodiment, the present invention contemplates the use of each of the antibodies individually or in combination, the antibody against p6 (INK4a), the antibody against Ki-67 and the anti MJC441 (anti-gene). H.sapiens DISC (Gene ID: 27185, NM_018662.2) Cancer-specific biomarker expressed by cervical cancer cells The combination of markers further facilitates discrimination between normal and cancerous cells proliferating and allows reproducible and standardized detection achieving a safe diagnosis that is performed at high performance for clinical samples of patients.

The integrated system also provides the ability to quantify the specific antigen-antibody reaction, in turn facilitating the automation of the cervical cancer detection process. In the present example, a colorimetric reaction is used, but other detection systems are encompassed by the present invention. Immunostaining based on colorimetric reading of cell reactivity it represents an accurate, cost-effective, easy-to-use analysis that is better suited to the visual assessment of staining intensities and the morphological examination of cells by the pathologist.

The present invention provides several improvements and useful applications thereof comparatively from the current methods of cervical screening: i) multiple unique high-throughput capacity, ii) increase the accuracy in diagnostic interpretation compared to the evaluation of cell morphology alone, due to the additional use of specific antibodies, iii) the use of specific and novel biomarkers indicative of the presence of human papilloma virus infection in cells of the cervix, indicative of the proliferative capacity of cells and specific for cancer cells, iv) the integration of robotic system that allows a high performance screening, and the centralized and integrated system makes all the diagnostic setting is made to a measure of cost-effective, easy to use and quantitative of the antigen-antibody reaction.

The characteristics of the present invention, and its applications to the detection of detection of cervical diseases at high performance will result in a grand benefit for the diagnosis of uterine cervical cancer, particularly in countries with low resources and infrastructures and in developed and developing countries.

DESCRIPTION OF THE FIGURES Figure 1 is a flow chart of sample processing / data data flow of the system of the invention.

Figure 2: Immunodetection of MJC441 in cervical cancer cell lines Left panel: Diagram illustrating the location of point 4 cervical cancer cell lines in the MPAT assay. The same amount of proteins from the extracts of each cell line were placed, in duplicate, in the MPAT membrane in a matrix of 6 columns and 4 rows, and tested with mAb MJC441. The proteins are from tissue culture supernatants with (s +) or without (-s) fetal calf serum (FCS), or from cell extracts (x), as indicated. The cell lines are: Ca Ski (a), ME-180 (b), C- 33A (c), and SiHa (d).

Right panel: MPAT immunodetection (described in Example 6) of MJC441 either secreted into tissue culture supernatants or expressed in cervical cancer cell lines, using mAb MJC441, as described in Example 4. Intensity of the point refers to the levels of expression.

Figure 3: Detection by immunohistochemistry MJC441 MJC441 mAb was used to detect tissue MJC441 cervical patient's neck by IHC using microareglos (microarrays) tissue (TMA). TMA clinical samples are summarized in Table 1. As described in detail in Example 5, TMA slides were treated for antigen retrieval, inactivation of endogenous peroxidase, also blocking endogenous biotin and non-specific binding. Slides were incubated with mAb MJC441, followed by biotinylated secondary antibody. Immunodetection is based on the reaction of streptavidin peroxidase followed by the chromogenic substrate AEC, and the counterstaining of hematoxylin.

Panels A and B: cervical cancer staining (A) and the normal adjacent tissues of the same patient (B) with the mAb MJC441, from the nucleus 24 of cervical cancer of TMA; Extension: 20X.

Panels C and D: staining of two cases of cervical cancer from different patients with mAb MJC441, from the 96 basic TMA; Extension: 20X.

Figure 4: Immunostaining of cervical cancer and mixing normal cell line with mAb MJC441.

Immunostaining is demonstrated in this document using a negative control mAb (A) and mAb MJC441 (B); Extension: 40X. In the image, red arrows point to normal cells, green arrows to cancer cells. As described in Example 6 a mixture of normal cells (fibroblasts from human embryos MRC5) cells and cell lines of cervical cancer CaSki or C33A, mixed in a ratio of 1: 1, and let stand ON to adhere to 37 ° C in 15 wells multi-test slides. The next day after permeabilization, the cells were reacted with the undiluted mAb MJC441 followed by immunodetection and hematoxylin as contrast staining described in Example 6.

Figure 5: Immunostaining of cervical patient samples with mAb MJC441 multi-well device Cervical cells of a patient sample in preservative solution are layered in a well device multiple wells of the present invention, fixed in 95% ethanol, dried in air and rinsed with PBS, then they fixed and were stained with mAb MJC441. Immunodetection and hematoxylin contratinction was performed as described in Example 7; Extension: 40X.

Description of the microarrays of human cervical cancer tissue (TMA) of 24 or 96 kernels used in this document. The 24 basic TMA includes the normal adjacent tissue counterpart of each tissue suffering from cervical cancer. The 96 core TMA core of 48 samples in duplicate, including cases of cervical cancer, and normal, benign (cervical polyps) and inflammation (chronic cervicitis) tissues of the cervix. All tissues were from surgical resection. Cervical cancer histological subtype is indicated.

DETAILED DESCRIPTION OF THE INVENTION The invention includes a package of integrated services from a kit to private physicians, health providers and hospitals. The kit typically contains a speculum, a brush and detailed instructions on how to collect the clinical samples, a conical polycarbonate tube with a preservation fluid to preserve the cells and infectious microorganisms to prevent their degradation. The kit also contains bar code labels or other unique patient identifiers attached to the patient's clinical sample (s) and for any of several data collection formats that collect clinical information of the patient and their physician or health care provider information. The present invention includes the collection and delivery of coded vials containing the clinical samples of the patients and the bar of coded data sheets of clinical information and patient doctors or information from the health provider to communicate the identifier and a sample to a centralized processing and high-performance screening center for the detection of the disease and its classification. Upon arrival, the coded samples and the clinical information of the patient, the doctor and the information of the private or public health provider are entered in a database at the centralized diagnostic detection center before processing. The integrated system comprises anguide for the collection and transfer of clinical samples from patients to conventional cytological staining and markers.

I property applied to the multi-well device for high throughput screening, including conventional haematoxylin or hematoxylin and (H &E) eosin staining, and conventional Papanicolaou staining.

Between each stage of the communication, that is, between the patient and the provider, the laboratory provider, and returning the patient and to provide a coded system can be used to preserve the patient's privacy by removing the patient's identity from the sample. In one such system the patient is provided with a key code that can not be accessed by the laboratory, and the detection is carried out anonymously until the results are returned to the provider associated with anonymous coded results for the laboratory with patient samples and their clinical and personal information. This coded process avoids all errors that may occur during the processing and thus ensures the anonymity of the patient and a safe diagnosis at the same time.

The system combines the clinical samples of the patient coded in the multiwell for the processing and analysis of multiple samples simultaneously. For parallel processing the system of the invention uses a "multi-well" device for high performance analysis and screening. The multiwell has a solid support with multiple well-separated circular areas, each with a capacity of a patient sample, which leads to the simultaneous evaluation of multiple patient samples and multiple disease markers.

Once the processing and analysis is completed, the individual data resulting from any of the tests performed are correlated for each patient individually and a patient-specific data report is generated for and sent to the destination from where the sample arrived. from the patient to the doctor or provider after decoding with a unique identifier.

See Figure 1.

The multi-well device comprises approximately 40 numbered (1-40) circular zones (or wells) of 15 mm in diameter arranged in a matrix. The number of wells in the array and thus the diameter of the assay format (e.g., the number of cells) can be altered according to the type of assay and the number of samples in the relevant cell-based assay.

The multi-well device is preferably made of polycarbonate, however, other materials are encompassed by the present invention, such as, but not limited to: glass, acrylic, plexiglass, polystyrene, polypropylene, etc. Modifications of device material with respect to facilitating the adsorption of proteins, or ensuring compatibility with aqueous or organic solutions of the process or other modifications as known to those skilled in the arts, are encompassed by the present invention.

The multi-well device is coated with a material such as a Teflon film or other materials known in the art to partition the surface into circular areas to avoid cross-contamination between the samples. As the layer can be of different thickness, the circular areas can be delineated by a edge of different thickness and height, turning the area into a well of different depth Referring to Figure 1, a high-throughput screening system for patient sample screening uses a coded vial containing the patient samples in preservative solution is received and placed in a 40-investment sample holder. A liquid handling robot transfers patient samples from the individual vials to a coded multi-well plate. When the samples are divided so that more than one will accommodate a sample from the same patient, the code applies to more than one agreed upon. Papanicolaou stain or other tests, including immunostaining using the combination of antibodies described herein, multiple staining vessels are carried out in wells. Several multi-well plates can be combined in a rack to increase performance, while a high-performance automated system moves the multi-well racks from one stain cell to another, allowing the multi-step staining process.

The system allows the analysis of multiple samples simultaneously. In so far, it has utility application as a high performance format for a variety of cell-based assays including the Conventional cytology and cell immunostaining is usually performed on individual glass slides. While different pathologies are contemplated herein, preferred embodiments of the present invention focus on cervical cancer. The apparatus of the components of the system are paired to the information processes and discrete steps to track and safely combine the patient samples through the consumption, treatment, analysis and reporting of the results back to the patient, the doctor or health professional. The flow of information starts with coded samples received with singular or patient identification encryption, and a list of requested tests, usually directed along with information from the health provider. These data are entered into the central database of the diagnostic screening center before processing. The unique identifier of the patient is linked to one or more wells of the multi-well map (row / column) for each relevant trial that will be performed for that patient. The data obtained as output for each patient and for each test format performed were entered and merged into the database, assembled into a patient report, and sent the information through a secure connection portal and encrypted for the patient, doctor or health service provider.

The cervical samples. Cervical samples are obtained from an individual patient or a patient's primary contact, typically a local healthcare provider. Samples are barcoded and optionally provided with a unique identifier that has a secure data key / lock or encryption identifier that matches the patient's secure identification to the sample. The sample is collected from a patient's cervix, either doctor or health professional collected, or self-collected, including cells, tissue or body fluid, using any device, including, but not limited to brush, sponge, spatula by scraping or rubbing an area or by using a needle to aspirate body fluids. Cervical specimens may also be derived from, but not limited to, fresh or frozen human tissues, paraffin-embedded tissues, biopsy specimens, exfoliated cervical cells, or samples from the cervical mucosa, or cervicovaginal wash samples.

The samples comprise cervical cells, in suspension, derived from cervical sample collected according to liquid-based cytology such as, but not limited to, the preparation of ThinPrep® (CYTYC, Inc.), or SurePath®.

(TriPath Imaging, Inc.). In fact, in particular embodiments of the present invention the cervical cells derive from ThinPrep aliquots.

The cell suspension of the cervix may use proprietary cytological preservative solution, another alcohol-based cytology or immunohistochemical solutions, or cells of the cervix suspended in non-preservative solutions, such as phosphate buffered saline (PBS).

With the system of the present invention, the samples can be processed in parallel and evaluated by cytological staining microscopy examination, as well as the combination immunostaining with specific antibody described herein.

High performance hematoxylin staining is demonstrated in (Example 1). Cells from a sample of the patient's cervix in preservative solution are deposited in a circular area of the multi-well device. Multiple samples are handled at the same time, stained and evaluated visually under the microscope.

The plurality of preserved samples is deposited in the multi-well device, which has first been UV irradiated to allow cell attachment. They are covered Other methods for cell attachment.

After depositing the conserved samples in the multiple wells, the cells are fixed in 95% ethanol and the entire multi-well device is air-dried at room temperature, or alternatively in a 37-40 ° C chamber, then Rinse with PBS. Cell debris is erased and hematoxylin staining is performed throughout the range. The matrix of the samples was visually examined under a microscope and the result reported for all the samples in the matrix.

Papanicolau staining of cervical samples of patients using the multi-well device. The system includes the staining of normal and abnormal cells in the multi-well device with any cytological stain, including the Papanicolau stain used in cervical cytology screening in combination with the biomarkers described above.

In this preferred form (Example 2), 50 samples of normal cervical dysplasia and 50 cervical patients in preservative solution, including, but not limited to, ThinPrep samples, the latter comprising low and high degree of dysplasia of and samples of ASCUS, they were processed by a simple centrifugation and the resuspension stage. Processing of the clinical sample included the decanting of the patient cervical sample in solution preservative and using 2 ml of the remaining solution to pellet the cells of the cervix by centrifugation at low speed. Samples were transferred to a block of 96 ml deep well 2.2 for high throughput processing.

Then, the cell pellet was resuspended in 95% ethanol, and a fraction of the resuspended cell pellet was manually deposited in the multi-well device, one patient sample per circular area. The samples were stained using Papanicolaou staining solutions and read by a blind pathologist at the initial diagnosis of the sample. The results of the diagnosis obtained by the present invention recognize normal, normal samples and samples of the disease as abnormal at very high rates.

It is understood that these patient cervical samples were collected either by a health professional or self-collected, either in the form of SurePath SurePath or samples, or of other substances as described above. Other sample processing is contemplated for sample concentration, purification, clarification of mucus, blood, and other cellular debris, other methods of sample enrichment and processing, including but not limited to the BD SurePath manual method by which a sample of the cervix is enriched after passage in a gradient density, centrifuged, resuspended and deposited in a single slide by sedimentation by gravity through a sedimentation chamber.

As used herein, the term "antibody" refers to a polyclonal, monoclonal, recombinant, full-size molecule or antibody fragment thereof, including, but not limited to, Fab, scFv, variable single-strand fragment , affibodies, diabodies, and any other antibody fragment retain the relevant antigenic binding site. The term "antibody" is used interchangeably herein to refer to any of the above species. Therefore, antibodies include antibodies produced in vitro, as well as antibodies generated in vivo in a species capable of immune response. Methods for producing recombinant antibodies, monoclonal and polyclonal fragments thereof are known to the person skilled in the art (Colligan et al, Current Protocols in Immunology, Wilcy Intersciences; Kohler et al., Nature 256: 495-497, 1975; phage display of peptides and proteins - A laboratory manual, BB Kay, Winter J. &McCafferty J., Eds, Academic Press, 1996).

Antibodies specific to biomarkers Specific tests can help in the diagnostic interpretation of preneoplastic lesions and dysplasia in the detection of cervical cancer, by identifying specific markers that are overexpressed in precancerous or cancerous conditions. Next, such a new marker is described. Immunostaining may serve as a complement to morphological interpretation by the pathologist, by providing greater accuracy to histopathology or diagnosis based on cytology.

MJC441 Marker .MJC441 is Homo sapiens gene DISC1 (Gene ID: 27185; NM_018662.2), which encode the altered protein in schizophrenia 1 (DISC1), composed of 854 amino acids, with predicted MW of 93,611 Da (UniProt Q9NRI5; NP_061132.2 ). This protein, which houses spiral coil motifs, is involved in the regulation of several aspects of embryonic and adult neurogenesis, including neurite growth and cortical development through its interaction with other proteins. A chromosomal aberration involving the DISC1 segregates genes with schizophrenia and related psychiatric disorders in a Scottish family. There are multiple known variants of alternative splicing and isoforms, especially in the brain (provided by NCBI Ref Seq; Pruitt, 2012).

Immunodetection of MJC441 in cervical cancer cells HPV-containing cells such as (Ca ski, ME-180 and SiHa) and non-HPV-containing cell lines such as (C-33) cervical cancer cell were not cultured in absent fetal calf serum to facilitate detection of markers potentially secreted in their culture supernatant. The immunodetection procedure is described in detail in Example 3. As exemplified in Example 4, protein extracts were prepared from cells and their cell culture supernatants, and equal amounts were seen in the MPAT, and were reacted with mAb MJC441, followed by detection of antigen-antibody complexes.

As illustrated in Figure 2, strong expression of MJC441 is observed in extracts of Ca Ski, ME-180 and SiHa cell lines, as well as in non-HPV containing C-33A. The secretion is observed mainly in the supernatant of the cell culture without fetal calf serum (lanes) of the culture of Ca Ski.

MJC441 expression in tissues of cervical cancer cancer tissues by immuno-skeletal Immunohistochemistry (IHC) is a common practice in in vitro diagnostic procedure used to determine the normal or disease status of a patient's biopsy. The patient's biopsy is first fixed in formalin and paraffin and embedded, then sectioned at 3-5 micrometers thick and mounted on treated microscope slides to improve tissue adhesion. The slides were stained with a relevant antibody against a cell marker in a procedure described in detail in Example 5. Tissue microarrays (TMA) can also be used in place of individual lamellae. TMA, as is known in the art, allows the analysis of the reactivity of an antibody, or the expression of biomarkers in a large number of patient samples that allow the establishment of biomarker prevalence in the population.

In the example detailed herein, the immunostaining procedure comprises the use of anti-mouse IgG biotinylated secondary antibody followed by streptavidin linked to horseradish peroxidase, finally followed by the addition of AEC substrate. Other immunostaining procedures known in the art are encompassed by the present invention. For example, protocols based on different detection and labeling systems, such as alkaline phosphatase, biotin-streptavidin, or fluorophores can also be performed with success within the scope of the present invention. On the other hand, while most tissues must undergo a pre-treatment to inactivate the endogenous peroxidase if peroxidase-based staining is used, such pre-treatment is not necessary when fluorescence-based imaging is used, and the protocol is modified accordingly.

Modifications and alternatives known to those skilled in the art are encompassed by the present invention, such as, but not limited to, the following: any method for making antigens that is more accessible to antibody binding can be used in the practice of the invention , including antigen retrieval methods known in the art, alternative screening and staining protocols (such as the use of the DAB substrate in place of AEC), alternative methods of endogenous biotin blocking, or blocking non-specific binding of substrate .

Proteins have different localization within the cell depending on their function: they can be secreted (such as growth factors, hormones, neuropeptides), present on the cell surface (such as glycoproteiries, glycolipids and receptors) or are produced within the cell (in the cytosol, or in particular, sub- cellular compartments such as the nucleus, the Golgi apparatus, the endoplasmic reticulum). The proteins can be localized to cellular structures through the use of the corresponding antibodies by a variety of techniques known to those skilled in the arts, which can be performed in suspension of mammalian cells or adherent cells, and which are described in FIG. (Current Protocols in Immunology, Wilcy Interscience, John E. Colligan et al.), Such as, but not limited to, immunohistochemistry, immunofluorescence (IF) using FACScan (FACS), flow cytometry (FC) and indirect IF , but also electronic microscopy and other imaging techniques that provide information about the location of new subcellular structures. For IHC, the specific staining of mAb against a marker can be located in either of the two nuclei, cell membrane or cytosol. The knowledge of biomarker localization is important in the design of diagnostic and therapeutic applications.

The association of MJC441 for cervical cancer is evidenced by IHC. Using mAb MJC441, the presence of the MJC441 marker is detected in the tissues of patients with cervical cancer compared to normal homologs, as described in Example 5.

To confirm preliminary IHC, mAb MJC441 was tested on a 24-core TMA comprising 12 cases of squamous cell carcinoma (Stage I TNM, grade I to III) and 12 cases of corresponding normal tumor (NAT) samples (ie , cancer and the normal sample of the same patient). Information from the clinical sample is summarized in Table 1. As illustrated in Figure 3 (panel A versus B), mAb MJC441 gave strong and specific staining of cancer tissues compared to adjacent normal tissues. Stromal incidental spot may be a consequence of the purified mAb.

Table 1 TMA 24 sections: 12 cervical cancer 12 NAT, Including: TMA 96 sections: 48 samples in duplicate, including mAb MJC441 was also tested on a TMA of 96 sections of wetsuits, including 39 cases of cervical cancer and 9 controls. As summarized in Table 1, the majority of cases were squamous cell carcinoma of stage TI and T2, with grades ranging from I to III, as well as carcinoma in situ, and few additional adenocarcinoma and adenosquamous carcinoma cases . Controls included normal (4), benign (3 cervical polyps), and signs of inflammation (2 chronic cervicitis). As illustrated in Figure 3 (panels C and D), a strong specific staining of cervical cancer was observed. mAb MJC441 consistently stained cancer cases with little or no staining of controls, demonstrating the prevalence of MJC441 in the population of cervical cancer patients.

The data presented in this IHC document on cervical cancer TMA confirm the usefulness of MJC441 as a marker of cervical cancer.

The inotination of cervical cancer and mixed with normal cell line with mAb MJC441 mAb MJC441 detects cervical cancer cells in a mixed cell population, an experimental condition imitating a sample of cervical patient in preservative solution. As described in Example 6, the normal fibroblasts of the MRC5 cell lines were mixed in a ratio of 1: 1 to the cells of a cervical cancer cell line. The cell mixture was incubated with mAb MJC441 as the primary antibody, then the antigen-antibody interaction was detected with a biotinylated anti-mouse secondary antibody followed by streptavidin conjugated with peroxidase, and, finally, stained with the DAB substrate.

As shown in Figure 4 (panel B), mAb MJC441 specifically stained cervical cancer cells within a population of normal cells, as evidenced by the brown precipitate formed by the DAB reagent. This contrasts with the negative control mAb (Figure 4, panel A) that produces no staining of the cells, whether cancer or normal. Notice the green arrows that point to the cancer cells, and the red arrows that point to the normal cells.

In general, mAb MJC441 is able to specifically discriminate and stain cervical cancer cells in a mixed population containing normal cells, an experimental condition imitating a cervical patient sample, such as a ThinPrep or other cervical specimen from the cervix. patient in preservative solution.

Immunostaining of cervical cells from patient with mAB antibody MJC441 using the multi-wells An aliquot of the patient cervical sample in preservative solution is first layered in a circular area of the multi-well device, fixed in 95% ethanol and air dried. The cell sample is then treated in a protocol analogous to an immunostaining of cells, as described in Example 7.

As illustrated in Figure 5, the MJC441 mAb specifically stained cervical dysplasia cells compared to normal cells. In general, these data show that the mAb MJC411 reacts with the marker MJC441 in cell lines total protein extracts for cervical cancer and is secreted from at least the Ca Ski cell line. mAb MJC441 spots the cancer tissues of cervix, but staining of normal cells homologous from adjacent tissues by IHC, with high prevalence of cervical cancer, as demonstrated by IHC in cervical cancer TMA. In addition, mAb MJC411 specifically stains cervical cancer cells within a population of normal cells in a cell immunostaining assay. Finally, mAb MJC411 stained cervical dysplasia cells of the patient in an immunostaining assay performed on the multi-well lamella of the present invention.

. MJC441 can be used as a standalone marker or in combination with the other commercial markers described herein in various diagnostic applications, including in IHC of patient's cervical biopsies, and immunostaining of cervical specimen cells from the patient in preservative solution, such as, but not limited to ThinPrep. mAb MJC441 is useful in immunostaining as an adjunct to traditional Papanicolaou staining for the detection of cervical cancer, as antibody staining facilitates, and improves reading and accuracy of the microscopic evaluation of cell morphology When immunostaining is combined with the high performance offered by the multi-well device, and its MJC441 mAb marker provide significant improvement for the detection of cervical cancer and detection in the setting of low to moderate clinical infrastructure.

As discussed above, immunostaining may serve as a complement to the morphological interpretation of patient cell samples by the pathologist. Immunostaining of multiple cervical samples can be performed simultaneously in the multi-well device using commercially available antibodies, commonly used, as well as antibodies against novel markers, such as mAb MJC441, as described below. The use of antibodies against antigens known to have different cellular locations, i.e., nucleus, cytoplasm or membrane, may further aid pathological diagnosis based on cytology or histology alone.

Immunostaining of cervical samples from patients using cocktail of antibodies in the multi-well device or in ELISA-type format. The invention also provides an immunostaining procedure based on a suspension of conserved cells derived from a human clinical sample, and a combination of antibodies reacted simultaneously and analyzed in the multi-well device or in a multi-well ELISA type format. The cell suspension is derived from a sample of the cervix, and the combination of antibodies detects cervical dysplasia, preneoplastic or neoplastic lesions in a cervical sample.

The system of the present invention includes parallel imunodetection of samples conserved and encoded in a matrix using antibodies against p6 (INK4a), antibodies against Ki-67, HPV, and any other infectious disease of the cervix, as well as antibodies against biomarkers that are over-expressed in dysplastic, preneoplastic or malignant cervical cells. The new biomarker MJC441 described in this document, which is differentially expressed in cervical cancer compared to the tissues of normal patients and mAb against the marker, is used by parallel processing in combination with anti-Ki-67 and anti -PL6 (INK4a) to detect abnormal cells of the cervix in a patient sample to improve the clinical information collected in a series of conventional cytology samples.

Immunostaining can be performed either in the multi-well device (Example 7) or in a conventional 96-well plate type ELISA format (Example When the immunostaining assay uses the multi-well device, the matrix of the samples of conserved patients is treated simultaneously with 95% ethanol to preserve the structural characteristics of samples, and dried in the air to allow the cells to bind to the surface of multiple wells during the procedure.

When the immunostaining is carried out in an ELISA-type format, the patient's cells are fixed with ethanol, then they can be treated either as adherent cells or in cell suspension. Note that when dealing with cells in suspension, each step of the procedure is followed by a gentle centrifugation step to ensure cells are collected in the bottom of the well.

The matrix of the samples are treated simultaneously with H202 in view of inactivation of peroxidase before antibody staining, if a peroxidase-based staining method is used. Then immunostaining with specific antibodies is performed.

The combination of primary antibody used here is anti-r? B (INK4a), anti Ki 67 and anti-MJC441, followed by secondary antibody conjugated with biotin, and streptavidin conjugated with peroxidase. The procedure of immunostaining optimally comprises the use of anti-mouse IgG biotinylated secondary antibody followed by streptavidin bound to horseradish peroxidase.

Other methods include immunostaining-alkaline phosphatase phosphates, fluorophores, or polymeric labeling systems (where a polymer structure is coupled to horseradish peroxidase, or another enzyme, as well as for the detection of a secondary antibody). Cervical cells should be pre-treated to inactivate endogenous peroxidase if peroxidase-based staining is used. Pretreatment is not necessary when fluorescence-based imaging system is used, and the protocol was modified accordingly.

When the immunostaining assay uses the multi-well device the peroxidase substrate can be the DAB precipitant or AEC substrates, to allow a new microscopic examination of the matrix of the patient samples. On the other hand, the ELISA type format requires a soluble substrate to allow the reading of optical density through a microplate reader. Indeed, the antigen-antibody complexes are visualized by the addition of a soluble substrate, such as, but not limited to tetra-methyl-benzidine (TMB). Immunoreactivity was obtained by measuring the optical density (OD) of the colorimetric reaction through a plate reader at the appropriate wavelength.

The advantage of the ELISA type format is to provide for the quantification of antigen-antibody reactivity by a colorimetric reading, rather than by a semi-quantitative visual reading based on the microscope examination, as when using the multi-well device.

To take full advantage of the information provided by the antibody staining cocktail, this invention encompasses the use of an antibody cocktail composed of both primary rabbit and mouse antibodies. Thus, The detection stage can use both anti-rabbit and anti-mouse · secondary antibodies, each linked to either peroxidase or alkaline phosphatase enzymes, thus allowing double or multiple staining with spectrally colorimetric or fluorescent chromophores.

The integrated nature of the system described in this document facilitates the reading of the microscope, handling and manipulation, microplate readers handling and manipulation, and any other sample inspection or quantitative instrumentation for handling, including semi- automated, and fully automated versions of the immunostaining processing cytological samples, taut or component of the reading.

The methods of the present invention are in vitro uses of the devices and systems for the detection of high performance and the detection of different pathologies, specifically gynecological disease in patient samples. The steps of the procedure consist of manipulation of ex vivo patient samples after collection, with various modes of testing and high-throughput processing and sample detection to detect pathologies, including inflammatory cells of autoimmune diseases, neuronal cells of neurodegenerative diseases and cervical cells. The methods use the integrated system for the collection of patient samples, the coding of samples for analysis and most of the results, analysis of biological property markers, specifically using a high-throughput centralized selection process, and notification of the encoded results that correlate with the source or identification of the patient's sample.

High-throughput screening methods use the parallel processing of multiple patient samples using a common set of diagnostic techniques, including a unique set of markers for the detection of the disease. To enable high performance detection, the process steps using the "multiwell" device comprising a solid support with multiple separate circular areas, each accommodating a patient sample, leading to the simultaneous evaluation of multiple samples of patients with a common set of biochemical markers for high-throughput screening and disease detection. The methods are capable of increasing efficiency using a robotic manipulation device that transfers patient samples from conical tubes coded for the multi-well device to ensure standardization of the process and to allow high-throughput screening and detection of the disease in the samples collected.

In addition to aspects of the system, the methods of the invention include a single information flow from the high throughput transformation allowed by the integrated system. The methods include the input of the results of the diagnostic information in an internal database and the delivery through the Internet portal to the doctors, health providers and hospitals of the source patient where clinical samples were collected ..

The analysis components of the novel methodology include the use of antibodies, preferably in combination, and including both commercially available and proprietary monoclonal antibodies (Ab) capable of binding to a marker polypeptide for cervical cancer. Detection of the marker or antibody is carried out in the high-throughput format system for the processing of large numbers of samples and is carried out in parallel with other pap smears or cytological tests. Therefore, the methodology is applied sample handling and processing, high-throughput screening of diseased cells, detection and management of the disease in an integrated platform.

The integrated high-throughput screening methodology is also comprised of the use of a new immunostaining format and sample processing equipment for patient samples using the multi-well device assisted with robotic fluid handling machines and tracking of bar codes and systems of integration and reporting of database to health care providers. The use of a patented combination of markers quickly and accurately distinguishes cells from cervical cancer cells Normal cervicals in a patient sample. On the other hand, a specific antibody also detects an antigen that is overexpressed in cervical cancer cells. Therefore, the present invention takes advantage of the use of these specific antibodies in a high-throughput format to identify cancer cells, which increases the diagnostic accuracy in the visual assessment of cell morphology by the pathologist.

Finally, the methodology contemplates the use of the combination of triple antibody against p6 (INK4a), anti Ki-67, and anti MJC441 (anti-gene H.sapiens DISC (Gene ID: 27185, NM_018662.2)) specific biomarker of cancer expressed by cervical cancer cells. The combination of markers further facilitates discrimination between normal and cancer proliferating cells and allows reproducible and standardized high-throughput detection of clinical patient samples.

EXAMPLES The following abbreviations are used throughout. ddH20: double distilled water; hr: hour, min: minutes; sec: seconds; ON: during the night, rpm: revolutions per minute; RT: room temperature.

Example 1: hematoxylin staining of cervical samples from patients using multiple wells device.

A fraction of a patient from the individual cervical sample in preservative solution (e.i. 250 microliters in volume, approximately 50-100,000 cells) is placed over a single -circular area of the multi-well device. Multiple samples can therefore be manipulated and evaluated simultaneously. The device has been previously irradiated UV to allow cell attachment. However, alternative methods for cell attachment can be used depending on the material the device is made of. Cells were fixed in 95% ethanol and air-dried either at room temperature or in a 37-40 ° C chamber, then rinsed with PBS. Clearing the upper cellular debris, the cells are ready for any cytological spot.

For hematoxylin staining, multiple wells device is rinsed with ddH20, and covered with a few drops of Mayer's weak hematoxylin solution for 2 min-1. Multi-well device is rinsed twice with tap or bluish H20 solution (Scott's tap water, or sodium or lithium carbonate solution to ensure alkaline pH) to stain the cell nuclei in blue, and once with ddH20. Mounting medium can be applied as appropriate for the preservation of slides.

Alternatively, the multi-well device is observe immediately under the microscope using different magnifications either as it is or air-dried.

Example 2: Papanicolau staining of cervical samples from patients using the multi-well device. 100 ThinPrep vials comprising 50 normal samples and 50 samples of cervical dysplasia, cervical or equivalent patient samples in preservative solution, were processed through a simple preparation method described below, deposited in the multi-well device of this invention, were stained with Papanicolaou staining solutions and read by a blinded pathologist for the original diagnosis of the samples. Clinical samples of cervical dysplasia make up: 16 LSIL, HSIL 16 and 18 ASCUS.

Patient's cervical samples in preservative solution were processed in the following manner. Briefly, the vials were decanted from most fluids and approximately 2 ml of the solution containing the remaining cell was transferred to a 96 ml deep well 2.2 block for high throughput screening processing. The block was centrifuged at 2000 rpm at 4 ° C for 10-15 minutes to pellet the cells, the supernatant was decanted and the cell pellet was resuspended in 95% ethanol.

Typically, a volume of 200 microliters is used, and adjusted according to the size of the granules.100 microliters was then seen in the multi-well device, using a pipette tip to slightly spread the sample over the entire circular surface, and the remainder is stored for further testing. The multi-well device was allowed to dry for 15 minutes before staining.

For Papanicolau staining, glassware of multiple wells is preferably used, and stained in shallow plastic containers of adequate size, using 60 ml of staining solutions, and 100 ml of wash solutions to allow even staining and thoroughly wash the surface of the device with gentle agitation. Staining ThinPrep staining solutions used (Hologic), although Papanicolau staining solutions from other sources are encompassed in the present invention, and was as follows: Distilled H20: 10 min; ThinPrep-nuclear nucleus: 5 min; dH20: 10 sec; ThinPrep Rinse Solution: 1 min; dH20: 30 sec; Blue bluish ThinPrep Solution: 30 sec; dH20: 30 seconds, 50% alcohol: 30 seconds, 95% alcohol: 30 seconds; ThinPrep Orange G: 2 min, 95% alcohol: 15 seconds, 95% alcohol: 15 sec; ThinPrep EA Solution: 4 min, 95% alcohol: 1 min, 95% alcohol: 1 min, 100% alcohol: 30 sec, 100% Alcohol: 30 sec: 100% alcohol: 30 sec; xylene: 30 sec : xylene: 1 min; xylene: 3 min. Multiple well plates were mounted with organic mounting medium and read by a pathologist. Alternatively, if plastic devices are used instead of multi-well glass, the stages of dehydration in 100% alcohol, and the xylene compensation steps are omitted, and replaced with rehydration steps, the use of several washes in the decrease in the strength of alcohol followed by a wash with distilled water, before for assembly with aqueous mounting medium.

Example 3: Micro array protein matrix teenology: The microarray protein matrix (MPAT) technology is a multiplex protein matrix immunoassay for the simultaneous analysis of multiple biological samples under the same conditions.

The solid support of a matrix protein matrix is composed of a different number of chambers or compartments of different sizes, depending on the scope of the investigation. In its simplest format, the MPAT consists of 96 cameras. The biological samples have been stained or printed (see below) in a matrix arrangement within each compartment on a nitrocellulose membrane. The same matrix of clinical samples, including normal and Sick, or the same matrix of protein extracts from different cancer cell lines is printed in each chamber. Each individual compartment is then coated with a different antibody (polyclonal, monoclonal antibody fragment, Fab, single-stranded, single-chain, affibodies, or any other recombinant version of conventional or combinatorial antibodies), and processed for the detection of complexes antigen-antibody.

Analysis of Protein Samples: Protein samples analyzed by MPAT can derive from fresh and frozen tissues, either normal or disease, even from cancer patients, benign or inflammatory diseases, and normal controls. Protein samples can be derived from cell cultures, cancer cell lines, and cancer cell supernatants, and even from microdissected cell types or from a given subcellular compartment. The protein samples can also be derived from the patient's sera or any other biological fluid of the patient, and prepared as described in the Examples below.

Impression of total protein extracts: Extracts of individual protein samples can either be deposited and stained manually or printed with a robotic system (Genomic Solutions Flexys, PBA Robotics, United Kingdom). Equal amounts of routine protein (250 mg or 1 mg / ml of a protein solution of cancer cells) of each sample are printed in a matrix format of the MPAT membrane, in duplicate or triplicate, as long as it is considered appropriate.

The membrane is then incubated for 30 min in 2% H202 (hydrogen peroxide) solution to inhibit the endogenous peroxidase present in the clinical samples, rinsed twice in Tris-saline buffer (TNE: 10 Tris-HCl pH 7 , 5.50 mM NaCl, 2.5 M EDTA) and then blocked for 30 minutes with a solution of 1% non-fat dry milk in Tris-saline buffer containing 0.1% (w / v) of Tween 20 (TNET).

Antibodies: Subsequently, each chamber or each sample matrix was covered with a primary antibody or primary combination of antibodies. Antibodies are routinely diluted appropriately in blocking solution, followed by 1 hour of incubation at room temperature with constant agitation. Blocking solution is TNET containing 1% nonfat dry milk or equivalent blocking solutions.

Detection of antigen-antibody complexes: The membrane was washed 5 times for 5 min each in TNET, then incubated for 1 h with antibodies Secondary to all or some of the primary antibodies conjugated with horseradish peroxidase (Roche) diluted 1: 10,000 in blocking solution. The membrane is then further washed 5 times, as described above. Antigen-antibody-anti-antibody complex reactivity was measured by chemiluminescence, using the SuperSignal West Dura Extended Duration Substrate (Pierce). The image is captured using a CCD camera (charge-coupled device; UVP model biochemical, CCD camera grade 0, with dark room designed for chemiluminescence, fluorescence and visible).

Alternatively, instead of a chemiluminescent detection system and based on an image acquisition system based on CCD camera, a fluorescent based system can be used, for example, incorporating the use of the Odyssey infrared image acquisition system Li -Cor. The MPAT protocol is then modified accordingly. Inhibition of peroxidase is not necessary. The membrane was rinsed twice in Tris-saline buffer, and then blocked for 30 minutes in Odysscy blocking solution (Li-Cor). Combination of primary antibody is appropriately diluted in Odyssey blocking solution, followed by 1 hour of incubation at RT. The membrane was washed 5 times for 5 min each in TNET to Then, they were incubated for 1 h with secondary antibodies labeled with a fluorescent dye (IgG-IRDye 800CW) diluted 1: 10,000 in Odysscy blocking solution. The membrane is then washed 5 times more, as described above. The antigen-antibody -anti-antibody complex is measured by direct infrared fluorescence detection. The intensity of each complex is captured as an image by scanning the membrane with Odyssey infrared imaging system in the 800 nm channel at 84 m resolution. Protocols based on different detection and labeling systems, such as alkaline phosphatase, biotin-streptavidin, and fluorophores as described can also be successfully performed within the scope of the present invention.

The following internal controls can be provided routinely: i) the same sample matrix is superimposed with buffer instead of the primary antibody, followed by the secondary antibody, thereby revealing the background of the secondary antibody (without antibody control) ), and ii) the same sample matrix is superimposed with pre-immune serum or non-secretory hybridoma or dilution buffer, followed by secondary antibody, which reveals the non-specific binding of mouse immunoglobulins.

Example 4: Immunodetection of MJC441 cervical cancer cells Cancer cell lines: the following cervical cancer cell lines were used: CaSki (human cervical squamous cell carcinoma cell line, known to contain an HPV16 genome integrated in about 600 copies per cell, as well as HPV18 related sequences) , ME-180 (human cervical carcinoma cell line, known to carry HPV DNA, with higher homology to HPV39 than to HPV18), SiHa (human squamous cell carcinoma cell line of the cervix, said to contain integrated HPV16 genome, in one and fifty-nine copies per cell) and C33 (human carcinoma cervical line, cells derived from the cervical cancer biopsy, negative for HPV DNA). HEL299 (embryonic lung fibroblasts) and MRC5 (14-week-old fetl cell line) were used as normal controls. All cell lines were cultured in the media according to ATCC recommendations.

Preparation of protein extracts from cancer cell lines: cancer cell lines (to the drawer of 107) are grown in culture according to the recommendations of the ATCC, with 10% fetal calf serum, 100 mg / ml streptomycin and penicillin up to 80% confluence, collected, washed twice with PBS, resuspended in phosphate buffer (pH 8.0) and broken in the following buffer: 50 mM Tris-HCl pH 7.5, 2 mM EDTA, 100 mM NaCl, 1 % NP40, and a 1 mM vanadate solution containing the following protease inhibitors: PMSF, aprotinin, leupeptin a, 2 and 4 mM, respectively. The cell lysate was centrifuged for 5 min at 14,000 rpm. The protein concentration of the cancer cell extracts is determined using a BSA standard from the BCA (bicinchoninic acid) protein assay reagent kit (Pierce, Rockford, IL) using a 1: 200 dilution of extract, and. A microplate reader (Vmax, Molecular Device) is used to read the absorbance at 570 nm. The stock solution of protein extracts is used at 1 mg / ml.

Preparation of cell culture supernatants: the tissue culture supernatants (TCS) were centrifuged to remove cellular debris and the supernatants were precipitated by the slow addition of 1-1.5 volumes of ice-cold acetone. The precipitation was carried out on ice or at -20 ° C for 1 hour. After 15 min of centrifugation at 4 ° C using pre-cooled rotors, the tubes are inverted to completely remove the supernatants. The pellets are centrifuged quickly to completely remove the last drops of the supernatant.

Finally granules were allowed to dry for 5-10 min under the hood, and resuspended in 2.5 ml of 50 mM Tris, pH 7. Samples were homogenized with sonicator, whenever necessary, and protein concentration was measured at through a BCA trial (see above). The samples were diluted to 1 mg / ml of working solutions.

To prepare TCS without fetal calf serum (FCS) to facilitate the analysis of potentially secreted proteins by immunodetection analysis of cancer cell lines (by MPAT or Western blot), the cells are cultured up to 70% confluence, the complete medium and replaced with medium without fetal calf serum (FCS) and cultured for 25 hours at 37 ° C. Cell culture supernatant without FCS is then precipitated as above.

MPAT: Protein extracts from cancer cell lines and from cell culture supernatants were printed with robots in the MPAT in duplicate and processed as described in Example A using the mAb of the present invention.

EXAMPLE 5: Staining of cervical cancer cells in tissue from cervical yew samples for immunohistochemistry. With the mAb antibody MJC441 To demonstrate the specificity of MJC441 and mAb MJC441, and its use in diagnostic applications in the histology of Cervical cancer, lamellae with tissue or fixation of the uterus are presented to the cervical cancer tissues and the tissues of benign patients, and the tissues of normal controls (paired, that is, of the same patient, or can be used in the following way.

Section of 5-microns of human cervical tissue fixed with paraffin embedded in paraffin or tissue microarrays are deparaffinized by each slide in the oven at 60 ° C for 30 min followed by immersion in three xylene baths for 5 min each. The slides were rehydrated by immersion in two 100% ethanol baths for 5 min each, and then in 95% ethanol, 70% ethanol baths for 3 min each, and finally soaked in water.

The endogenous peroxidase was blocked by lamellar treatment with 3% hydrogen peroxide soon in PBS for 10 min at RT, then washed twice in PBS for 3 min each. The recovery of the antigen is obtained by heating slides in a pressure cooker with full pressure for 5 min in 10 mM Tris, 1 mM EDTA pH 9, or in 10 mM sodium Tris-citrate, 0.05% Tween 20, pH 6. The slides were then cooled to room temperature in the same buffer for 10-20 min, rinsed in tap water for 3 minutes, and lastly, submerged in Tris buffer.

To block endogenous biotin, which may be a problem in some tissues, the slides were incubated for 15 min at room temperature in a soon of streptavidin in PBS (100 g / ml), washed with Tris buffer, followed by incubation with a Biotin soon (500 mg / ml) in PBE (PBS with 1% BSA, 1 mM EDTA, 1.5 mM NaN3, pH 7.4) for 30-60 min at RT, and washed in PBS. The non-specific binding was further blocked by treating the slides for 15 min at RT in 3% horse serum did in PBE.

The slides were incubated with mAb MJC441 as the primary antibody (either supernatant of the undid cell culture, or 01: 02- 1:20 appropriately did in buffer PBE) for 30 min at 37 ° C, or 1 hour at RT or overnight at 4 ° C in a humidity chamber, then rinse 3 times for 5 minutes each in Tris buffer. The slides were covered with a 1: 1000 dion of biotinylated secondary antibody in PBE buffer, and incubated for 30 min at 37 ° C or 1 hour at RT, then washed 3 times for 5 min each in Tris buffer. The slides were then covered with a 1: 1000 dion of streptavidin conjugated with peroxidase did in PBE (without azide), and incubated for 30 min at 37 ° C or 1 hour at RT, then they were washed 3 times for 5 min each in buffer Tris.

Finally, a few drops of AEC substrate soon (1 ml of 4 mg / ml of AEC stock soon in DMF, plus 15 ml of 0.1 M Na acetate pH 5, and 15 milliliters of hydrogen peroxide 30%) are used to cover the lamellae. The reaction is allowed to continue for 10-40 min, then visualized under the microscope, and stopped with normal water whenever appropriate. The slides were rinsed in water, and in contrast to a few drops of Mayer's weak hematoxylin soon for 1-2 min. The slides were immersed in a 0.1% sodium bicarbonate soon until the nuclei turn blue. The slides were covered with aqueous mounting medium, placed in an oven at 70 ° C and then allowed to dry for 10-20 min or overnight at RT.

EXAMPLE 6: Immunostaining of cancerous and normal cervical neck cells with mAb MJC441 A mixture of normal cells (MRC5 human embryo fibroblasts) and cells from cervical cancer cell lines CaSki or C33A, were mixed in a ratio of 1: 1, and let stand overnight to stick to 37 0 C in 15 wells multipurpose slides. The next day, the cells were rinsed with PBS, fixed in 95% ethanol and then permeabilized in Triton 0.2% in PBS for 5 min at RT. To avoid non-specific binding, the cells were incubated with a solution of PBE (PBS with 1% BSA, 1 mM EDTA, 1.5 mM NaN3, pH 7.4) before the addition of primary antibody.

The cells are then reacted with undiluted mAb MJC441 for 1 hour at 37 ° C followed by incubation with goat biotin AffiniPure-SP-conjugated anti-mouse IgG (H + L; 1 g / ml in PBS) as secondary antibody . Immunoreactivity was detected by incubation with 40-50 ml of streptavidin peroxidase (1 mg / ml in PBS), followed by staining with DAB substrate. Hematoxylin was used to counterthe nuclei of cells in blue.

Example 7: Immunostaining of cervical samples from patients with mAb MJC441 or with coctail of monoclonal antibodies Cervical samples of patients in preservative solution (250 ml) are superimposed on the circular areas of the multi-well device. The cells were fixed with 95% ethanol, dried in air to ensure adherence, and rinsed with PBS. Note that cells must be treated with H202 to inactivate endogenous peroxidase, if a peroxidase-based detection system is used. To block the endogenous biotin cells are incubated for 30 min at room temperature in a solution of streptavidin in PBE without azide (100 mg / ml), followed by a PBST solution. Then, the cells were further incubated with a solution of biotin (500 mg / ml) in PBE (PBS with 1% BSA, 1 mM EDTA, 1.5 mM NaN3, pH 7.4) followed by three washes with PBST the cells are then centrifuged and the supernatant discarded.

The cells were then treated for immunostaining with mAb MJC441 or antibody cocktail (undiluted or appropriately diluted in PBS with 1% BSA, 1 mM EDTA, 1.5 mM NaN3, pH 7.4), enough to cover the cells. The primary antibody or a primary antibody cocktail was incubated for 1 hour at 37 ° C. Then, for detection, a biotinylated anti-mouse IgG secondary antibody (Jackson Lab.) Diluted to 1 mg / ml in PBE is added to, and were incubated for 30 min at 37 ° C. The secondary antibody was followed by streptavidin bound to horseradish peroxidase (Jackson Lab.) diluted to 1 mg / ml in PBE, and incubated for 15 min at RT.

The reaction was developed by the addition of a freshly prepared peroxidase substrate, such as AEC (3-amino-9-ethylcarbazole substrate) followed by incubation at RT. The color development is examined under the microscope and the reaction was stopped with PBS containing 0.05% azide.

Hematoxylin contratinction is performed for nuclei of colored cells in blue, and the multi-well device is observed under the microscope.

Example 8: Immunostaining of the cervical sample from a patient with a single antibody or a cocktail of antibodies using an ELISA-type format. This example describes how immunostaining can be performed using commercially available and commonly used monoclonal antibodies, as well as new antibody label of the present invention, or any other combination thereof. This example therefore further demonstrates the utility of immunostaining as a complement to the microscopic evaluation of cell morphology in cervical screening. In addition, the ELISA type format allows quantification staining.

In this example, the concept of multi-well device format extends to the ELISA format of commonly known type, such as a 96-well flat bottom plate or equivalent.

This assay involves cells -adherent or cells in suspension. 96-well tissue culture plates are preferably treated for cell adhesion, unless the protocol is used in the cell suspension mode, as described below. Note that When dealing with cells in suspension, each step of the procedure is followed by a gentle centrifugation step to ensure cells are collected in the bottom of the well.

Finally, the antigen-antibody reactivity can be quantified by colorimetric reading, instead of by a semi-quantitative visual reading based on microscopic examination.

In this embodiment, an aliquot (250 ml volume) of each patient sample of the cervix in preservative solution, such as, but not limited to, a Thin Prep is deposited in each well of a 96-well plate. The cervical cells are treated to inactivate the endogenous peroxidase with an appropriate volume of 1% hydrogen peroxide solution diluted in PBS (200 ml / well) for 30 min at RT with gentle shaking at 400 rpm. After inactivation of peroxidase, the cells are washed three times with PBS, centrifuged and the supernatant discarded.

At this point, the cells were resuspended in 95% ethanol for a fixation step and air-dried at room temperature to promote cell adhesion to the well. Alternatively, the cells were centrifuged gently at each step, if treated as cells in suspension.

To block the endogenous biotin cells are incubated for 30 min at room temperature in a streptavidin solution in PBE without azide (100 mg / ml), followed by a PBST rinse. Then, the cells were further incubated with a solution of biotin (500 mg / ml) in PBE (PBS with 1% BSA, 1 M EDTA, 1.5 mM NaN3, pH 7.4) followed by three washes with PBST the cells are then centrifuged and the supernatant discarded.

The plate is ready for immunostaining treatment. All incubation steps are 30 min long and performed at RT under agitation 400 rpm for the rest of the procedure.

The plates were incubated with the corresponding primary antibody, which may be either a single antibody or a cocktail of antibodies, i.e., a combination of antibodies, which may include commercially available commonly used antibodies, and / or antibodies against novel biomarkers such as It is described. For the purposes of this embodiment, the anti-p6 (INK4a), Ki-67 and MJC441 are used as primary antibodies, added to the cells (50 ml / well of an appropriate dilution in PBE buffer) and incubated for 30 minutes. min to TA. The cells are washed three times with PBST to remove the unbound primary antibody (specifically, the wells are filled with buffer, the plates were centrifuged 10 min at 1500 rpm, and the supernatant discarded) A secondary antibody conjugated with biotin (for the purpose of the invention, a goat anti-mouse IgG antibody in biotin conjugate) is then added to each well (50 ml of a lpg / ml solution of the biotinylated secondary antibody in PBE buffer). ) and the cells were incubated as described above (30 min, RT, 400 rpm). The cells are washed three times with PBST to remove excess secondary antibody as described above. The cells were centrifuged for 10 min at 1500 rpm, and the supernatant was carefully aspirated, preferably with vacuum.

Streptavidin conjugated with peroxidase is added to each well (50 ml of an lpg / ml dilution in PBE buffer without azide) and the cells are incubated 15 min at RT with shaking at 400 rpm, as described above. After a wash with PBST and two washes with PBS, the antigen-antibody complexes are visualized by the addition of 50 m? of TMB substrate solution to each well, followed by incubation for 10-40 minutes at room temperature and stirring 400 rpm. The blue color will appear in 10 minutes with variable force based on the number of cells per well and on the concentration of antigen. The reaction stop by filling wells with H2S04, turning the blue solution to a yellow color that reads at 450 nm. Immunoreactivity was scored using a colorimetric ELISA plate reader. Alternatively, after a rapid turn, the supernatant was carefully transferred to a fresh plate and read immunoreactivity.

For quantification purposes, controls should be included such as cells from a normal cervical sample with the primary antibody, and cells from each patient sample with and without the primary antibody, equivalent number of cells, etc.

Claims (11)

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following is claimed as property CLAIMS

1 . A high throughput system for processing patient samples for cervical cancer screening comprising: a plurality of cervical samples of patients preserved in solution, wherein the plurality of individual patient samples containing the cervical cells are deposited in individual wells of a multi-well device, where one well contains the cervical cell samples of a single patient in the multi-well device, and where the cells of the cervix are kept in a liquid cytology preservative solution; apparatuses and reagents for cytological staining of the plurality of patient samples by simultaneously exposing the plurality of individual wells of the multi-well device to a cytological spot; a combination of antibodies specifically reactive with cervical cancer markers immunologically react with the patient's sample; apparatus to examine microscopically the cells of the cervix are cytologically stained and reacted immunologically to determine the presence or absence of abnormal cells

2 . The system of claim 1, wherein the cytological hematoxylin stain or is Papanicolau stain.

3 . The system of claim 1, wherein the system further comprises an automated apparatus for cytological staining.

Four . The system of claim 1, further comprising electronic means for matching the patient sample with a patient identification.

5 . The system of claim 1, wherein the combination of antibodies includes anti-pl6, anti-Ki-67 monoclonal antibodies, anti-MJC441 antibodies or combinations thereof.

6 An in vitro method for high processing performance of patient samples, comprising: ensuring preserved patient samples having each encoded identification and scanning to the patient; depositing a plurality of patient samples in a multi-well device, in which one cell individual of the multi-well device is identified with a single patient sample; which simultaneously expresses samples from patients with cytology to identify abnormal cells in patient samples; immunologically reacting each patient sample with a plurality of antibodies; Y microscopically examining patient samples for the presence or absence of abnormal cells.

7 The method of claim 6, wherein the step of exposing patient samples or cytological spots comprises exposing the cells in the hematoxylin or pap smear sample.

8 The method of claim 6, wherein the method is further composed of the automated manipulation of the multi-well device during cytological staining, immunological reactions, or microscopic examination

9. The method of claim 6, further comprising introducing coded patient information into a database and electronically matching results of the microscopic examination of a patient identification. 10 The method of claim 6, wherein the Immunological reaction consists of the exposure of patient samples to a combination of antibodies comprising anti-MJC441, anti-p6, or anti-Ki-67 and combinations thereof. eleven . The method of claim 10 further comprising the step of reacting a secondary antibody with either anti-Pl6, anti-Ki-67, or anti-MJC-44. SUMMARY OF THE INVENTION The present invention describes a new device and in vitro methods of application thereof with utility in the detection of high performance, detection and treatment of disease diseases, especially gynecological and cervical infertility. The "multi-well" device of the present invention consists of a solid support with multiple well-spaced areas, each accommodating a patient sample, which leads to the simultaneous evaluation of patient samples. The methods of the present invention comprise conventional cytological staining or Papanicolaou staining of the cervix, and immunochemical staining using antibodies or combination of antibodies that are capable of binding to biomarkers that are overexpressed in cancer including in cervical carcinoma and dysplasia, compared to normal controls. The device and methods of the present invention can be practiced in any of the manual or automated modes, and applied to any biological fluid or cell suspension of any biological sample in view of a variety of cell biology assays, and in view of of detection and detection of cervix and other diseases. The present invention describes a new device and the methods of application of the same with utility in the detection of high performance, detection and treatment of the disease of cervical disease. The "multi-well" Device of the Present Invention consists of the UN Solid Support with Multiple Well-separated Areas, Each Accommodating a Patient Sample, Which Leads to the Simultaneous Evaluation of Patient Samples. The Methods of the Present Invention