RELATED APPLICATIONS

This application is a national phase application of International Application No. PCT/IN2019/050540, filed on Jul. 24, 2019, which claims the benefit of Indian Patent Application No, 201811028087, filed on Jul. 26, 2018. The entire contents of these applications are hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates to a screening kit for detection of different grades of cervical cancer. More, particularly the present invention relates to efficient platform developed to identify pre-cancerous lesions of the cervix viz.: (high-grade squamous intraepithelial lesion (HSIL) and cervical squamous cell carcinoma (CSCC) in the cells exfoliated from uterine cervix using a differential Raman spectral pattern.

BACKGROUND OF THE INVENTION

Cancer of the uterine cervix is one of the most common cancers among women worldwide and the second most prevalent cancers among women in India. It is considered as a preventable cancer as cervix is an easily amenable organ and occurrence of a heterogeneous spectrum of epithelial abnormalities (precancerous lesions) 10-15 years prior to the occurrence of invasive cancer has been well established (Sujathan et. al). The significance of Pap smear test for the detection and eradication of these precancerous lesions of cervix has been well documented. Based on this, systematically organized screening programmes for cervical cancer have been implemented in many of the developed countries. A rapid reduction in the incidence of this disease has been observed in countries like Sweden, Demark, Finland etc, where women have been covered by organized Pap smear screening programmes. The role of persistent infection with high risk HPV in cervical carcinogenesis is now well established and prophylactic vaccine against two of the high risk HPVs are currently available. Considering the several other high risk strains of this virus prevalent among the women, vaccine plus Pap smear test and HPV DNA test is now advocated for the effective control this disease. However, in India and many other low resource countries none of these measures have been implemented so far and cervical cancer continues to take extraordinary toll on the lives of our women. India contributes one fifth of the global burden of cervical cancer. As per the Globocan report of cervical cancer, around 528,000 estimated new cases with 266,000 death cases have been reported in 2012. Almost 9 out of 10 cervical cancer deaths occur in low resource countries including India. If we could also introduce population screening programmes, we could have saved the lives of our women. The major impediment for implementing screening programme by Pap smear is the lack of trained cytologists for microscopic analysis of the Pap smears of the eligible women of the community. So there is an urgent need for alternative cost effective and reliable method of screening without the requirement of highly trained cytologists.

Pap smear test is the microscopical analysis of cells which are naturally exfoliating from the squamocolumnar junction of cervix. These cells often stick to the cervical mucus which is collected by scraping with a spatula. These cells are studied for various morphological features of precancerous lesions (LSIL, HSIL etc) or malignancy. Since the precancerous lesions are asymptomatic, only through regular screening programmes it can be picked up. The microscopic analysis of cells require highly trained cytotechnologists. The doctor at the time of examination, visualize the cervix and collect a cell sample from the cervix and preserve in a preservative fluid. These cells are smeared onto a glass slide, stained with Pap stain and studied under microscope by trained cytotechnologists for morphological features and reported as normal or positive for precancerous lesions LSIL/HSIL or positive for malignancy. All positive samples are further confirmed by a cytopathologist. The smears are often reported as per the modified Bethesda system. The precancerous lesions are treated and followed up in a systematic way to ensure that the lesion is not progressing. As per the reports of the developed countries, where there is regular systematic screening program using Pap smear, more than 85% of the smears are within normal limits. So, if these normal smears can be screened out by a cost effective method, without utilizing the skilled cytotechnologists, the other 15% can be subjected to manual methods without over use of the resources.

Because skilled cytotechnologists are less available and as it is a tune consuming technique, some other alternative techniques are in high demand. The demand for specific and accurate cervical cancer screening has driven the development of novel diagnostic probes having high selectivity and sensitivity. Raman spectroscopy (RS) based on the inelastic light scattering can provide important biochemical information of macromolecules such as proteins, nucleic acids and lipids, because each molecule has its own pattern of vibrations that can serve as a Raman signature. Raman spectroscopy recently has emerged as a novel non-invasive diagnostic tool for cancer detection and identification of malignancy at different stages of neoplasia in tissues. Some groups have investigated the applications of Raman spectroscopy in differentiating normal and malignant tissues in various body sites, such as lungs, stomach, bladder, breast, parathyroid, prostate and cervix.

However, Raman scattering suffers the disadvantage of extremely poor efficiency due to its inherently small cross-section. Besides, the Raman spectra of biological samples are often superimposed on top of a strong fluorescence background that may be huge and make it difficult to extract the Raman signals. So in medical diagnosis these disadvantages make it a great challenge for practical applications of conventional Raman spectroscopy. There has been a great interest in Raman spectroscopy technique in the past two decades owing to the discovery of the surface-enhanced Raman scattering (SERS). Surface-enhanced Raman scattering was first reported by Fleischman et al. in 1974. Recent studies show have shown that with SERS technique, Raman signals can be enhanced by 10 to 14 orders of magnitude when the Raman active molecules are attached to nano-textured metallic surfaces, simultaneously with reduced autofluorescence background. SERS technology greatly improves the detection sensitivity of Raman spectroscopy, and has drawn considerable attention due to its great potential in biomedicine. SERS based immunoassay, which relies on a specific interaction between an antigen and a complementary antibody, is the strategy developed for most current oncology applications. Label-free SERS produces huge and complex data sets and it necessitates more refined analytical processes to mine substantial information from the spectral data. A great inconsistency in the intensity and spatial scattering generated due to the variable deposition and orientation of gold nanoparticles reduces the clarity of spectral data obtained. Even though such challenges are existing, introduction of chemometric analysis like Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA), Cluster Analysis, Support Vector Machine Analysis (SVM) have enriched or improved the spectral data sets for classification and characterization.

For cervical cancer, smears made from scraped cervical exfoliated cells is the accepted screening method which can be taken conveniently and even continuously throughout the treatment for diagnosed patient without causing pain in a non-invasive manner and biopsy is done for confirmation of high grade lesions and invasive carcinoma. All studies reported earlier used tissue samples for Raman measurements rather than cell samples. Collection of tissue sample needs surgical procedures and hence cannot be suggested for screening purpose. Gold nanoparticles (AuNPs) are used in this study as SERS substrate because of their favorable physical, chemical properties and biocompatibility.

In this invention, we explored the use of AuNPs for SERS spectral evaluation in cervical exfoliated cells in order to find out the biochemical changes which could able to differentiate between NRML, HSIL and CSCC patients by distinct changes in Raman fingerprints of variable abundance of intracellular amino acids with associated proteins, lipids, nucleic acids, DNAs and variables viz., amide II, amide III etc. To our knowledge, this is the first report on SERS based analysis of cervical exfoliated cells using a label free nanoformulation with AuNPs for cervical precancerous detection. The results showed significant level of differentiation between three classes using single cell, cell pellet and extracted DNA measurements.

Definitions

AuNPs—Gold nano-particlesNRML—NormalHSIL—high-grade squamous intraepithelial lesionCSCC—cervical squamous cell carcinoma

