The Nuclear Changes Characteristics of Papillary Carcinoma - Case Study Example

Methods Used in Biochemistry s Affiliation Introduction Thyroid cancer is the most common endocrine malignancy with more deaths annually than other endocrine cancers combined. Thyroid cancer incidence is elevating throughout the world. Studies indicate that this rise is due to increase papillary carcinoma, the most prevalent thyroid malignancy in areas with insufficient iodine. Incidence rates annually vary by geographic area, age and sex (Rego-Iraeta, Pérez-Méndez, Mantinan, & Garcia-Mayor, 2009). A normal fully differentiated thyroid follicular cell is transformed to a rapidly growing undifferentiated anaplastic thyroid carcinoma cell involves a number of stages through growth stimulation and mutagenesis. Pathological features include papillary carcinoma and follicular carcinoma. Papillary carcinoma is an encapsulated tumour with papillary and follicular structures that is characterised by overlapping cell nuclei that have a ground glass appearance and longitudinal grooves with invaginations of cytoplasm into the nuclei. Follicular carcinoma by follicular differentiation but without the nuclear changes characteristics of papillary carcinoma (Rego-Iraeta, Pérez-Méndez, Mantinan, & Garcia-Mayor, 2009). The first symptom is usually lymph-node metastases. Other symptoms include hoarseness, dysphagia, cough and shortness of the breath in the advanced stage. The carcinoma is usually single and firm and moves freely during swallowing on physical examination. If the thyroid nodule is hard and irregular and the ipsilateral lymph nodes are enlarged, and there is a history of progressive increase in the size of the nodule carcinoma should be suspected. The best method for distinguishing between malignant and benign thyroid nodules is fine needle aspiration cytology. Thyroid ultrasonography is useful for assessing the size of the nodule, detecting other nodules and guiding fine needle biopsy if a nodule is difficult to palpate or small (Rego-Iraeta, Pérez-Méndez, Mantinan, & Garcia-Mayor, 2009). Surgery can be used to remove tumour from the neck by resecting the affected thyroid gland and affected cervical lymph nodes. Other treatments include, Iodine-131 therapy and external radiotherapy. Follow up should be done so as to maintain adequate thyroxin therapy and to detect persistent or recurrent thyroid carcinoma (Schlumberger, 2000). Materials and methods Enzyme linked immunosorbent assay (ELISA) was used to measure levels of five serum proteins as formerly shown to be up regulated in papillary thyroid cancer, that is, angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19) tissue inhibitor of metalloproteinase-1 (TIMP-1), galectin-3 and chitinase 3 like-1 (YKL-40). Serum levels were compared between patients with papillary cancer and those with benign tumours in order to identify serum biomarkers of papillary thyroid cancer (Savin, Cvejić, Mijatović, & Simonović, 2010). To detect immune responses in the body such as infectious microbes, ELISA test uses components of the immune system and chemicals. The tests usually involves antigens and antibodies. Purified human thyroglobulin could be prepared from surgical specimens of non-toxic goitre by homogenisation and column photography. Measurement of thyroglobulin measurement is basically used to monitor patients with differentiated thyroid carcinoma for tumour to re-occur (Savin, Cvejić, Mijatović, & Simonović, 2010). ELISAs rely on specific interaction between an epitope, a small linear or three dimensional sequence of amino acids found on the antigen and a matching binding site. The antibodies can be monoclonal or polyclonal. ELISA for human thyroglobulin antibodies (hTg-Ab) and microsomal antibodies (M-Ab). (Savin, Cvejić, Mijatović, & Simonović, 2010). Serum Tg principally integrate three variables: the mass of the thyroid tissue, the degree of thyrotropin (TSH) receptor stimulation and tumour’s intrinsic ability to synthesise and secrete Tg. The measurement of serum Tg should be interpreted relative to the TSH status of the patient. When the TSH is low, basal serum Tg may be undetectable and recombinant human thyrotropin administration may be needed to raise serum Tg into the range that can be measured (Savin, Cvejić, Mijatović, & Simonović, 2010). Sera from prostatic cancer patients contain anti-idiotypic antibodies is indicated by experimental results observed in connection with binding of monoclonal antibody 7E11-C5 detected in competitive inhibition ELISA. There is an association between persistence of tumour and the presence of circulating anti thyroglobulin antibodies in patients with thyroid cancer (Savin, Cvejić, Mijatović, & Simonović, 2010). Screening for monoclonal antibody production and general enzyme immunoassay of antigen is done by enzyme linked immunosorbent assay (ELISA). ELISA measures the concentration of an analyte in solution. The separation of specific and non-specific interactions occurs via serial binding to a solid surface. ELISAs are rapid and easy to carry out since they are designed to quickly handle a large numbers of samples in parallel and are majorly used for evaluation of various research and diagnostic targets (Savin, Cvejić, Mijatović, & Simonović, 2010). The first step of ELISA begins with coating with either an antigen or an antibody which is adsorbed to a polystyrene 96 well plate. Coating is followed by blocking and detection steps. Several washes are repeated between ELISA steps to remove unbound materials since the assay uses surface binding for separation. The excess liquid is removed in this process so as to prevent the dilution of the solutions added in the next stage. ELISAs can be complex including various intervening steps and the ability to measure protein concentration in heterogeneous samples. Human thyroglobulin adsorbed to polystyrene micro plates, human serum and anti-human IgG antiserum conjugated to alkaline phosphatase is used (Savin, Cvejić, Mijatović, & Simonović, 2010). In the TgAb kit, autoantibodies are placed in calibrators and controls and the serum from the patient are allowed to interact with Tg coated onto ELISA plate wells. Incubation is done after 15 minutes and samples are discarded leaving TgAb bound to the Tg that is immobilised. Protein A-Alkaline Phosphatase conjugate binds to the TgAb bound to the immobilised Tg in the second incubation step. In the third incubation step, the amount of protein A-Alkaline Phosphatase conjugate bound to the plate is determined by addition of p-nitrophenyl phosphate resulting to formation of the yellow colour. A stop solution is added to terminate the reaction and the absorbance of the yellow mixture is then read at 405nm using ELISA plate reader. A higher absorbance would indicate presence of TgAb in the test sample (Latrofa, et al., 2008). In other ELISA kits, autoantibodies are laced in calibrators and controls and patient serum are allowed to interact with Tg coated onto ELISA plate wells. The samples are incubated for15 minutes then discarded leaving TgAb bound to the immobile Tg. Protein-A-Alkaline Phosphatase conjugate is bound to the TgAb that is bound to the immobilised Tg in the second incubation step. A yellow colour is formed when p-nitophenyl phosphatase is added in the third incubation step to determine the amount of protein A-Alkaline Phosphatase conjugate bound to the plate. The reaction is stopped by the addition of stop solution and absorbance of the yellow solution at 405nm using ELISA plate reader. A higher reading indicates the presence of TgAb in the test sample (Latrofa, et al., 2008). ELISA can be used to analyse serum specimens according to manufacturers’ instructions. Chemokines and chemokine receptors are expressed in human papillary thyroid cancer derived cell lines. These receptors can be detected by ELISA. Normal thyroid cells were used a negative control. These expression levels can be evaluated later by Q-RT-PCR. Assay performance is directly related to the efficacy of conjunction and the quality of the detector and the antibody used for enzyme labelling (Latrofa, et al., 2008). Results Serum levels of four of five proteins were elevated in patients with thyroid masses relative to normal values. There is no statistically difference when comparing biomarkers of benign and papillary thyroid cancer. However Gal-3 and TIMP-1 display a greater potential difference. The biomarkers are directly related to clinical staging. The ELISA assays determine the quantity of the midkine and the pleiotrophin in thyroid tissue collected from fine needle aspirates. Normal levels of thyroglobulin antibodies have range at an absorbance of 405nm. Readings below or after indicate abnormality (McLeod, et al., 2014). Discussion Thyroid cancer is a common endocrine malignancy and an increasing malignancy in the world. Proper diagnosis is vital therefore thyroglobulin antibodies can be detected by ELISA test. Application of ELISA is used to detect autoantibodies in thyroid cancer in serum. Thyroglobulin autoantibodies (TgAb) are important markers OF thyroglobulin in the follow up of differentiated thyroid carcinoma. Tg measurement serves a biochemical marker of persistent or recurrent disease in thyroid cancer. Thyroid autoimmunity is important in determining prognosis of the disease (McLeod, et al., 2014). Quantification of serum tumour markers has an important role in determining whether the patients treated for cancer require therapy. This facilitates early detection, optimisation of methods for quantifying known tumour markers to improve management of malignancies. However ELISA is has limitations such as potential interference from endogenous immunoglobulins and imperfect concordance across platforms (Hoofnagle, Becker, Wener, & Heinecke, 2008). References Hoofnagle, A. N., Becker, J. O., Wener, M. H., & Heinecke, J. W. (2008). Quantification of Thyroglobulin, a Low-Abundance Serum Protein, by Immunoaffinity Peptide Enrichment and Tandem Mass Spectrometry. Clinical Chemistry, 1796-1804. Latrofa, F., Ricci, D., Grasso, L., Vitti, P., Masserini, L., Basolo, F., . . . Pinchera, A. (2008). Characterization of Thyroglobulin Epitopes in Patients with Autoimmune and Non-Autoimmune Thyroid Diseases Using Recombinant Human Monoclonal Thyroglobulin Autoantibodies . The Journal Oof Clinical Endocrinology & Metabolism. McLeod, D. S., Cooper, D. S., Ladenson, P. W., Ain, K. B., Brierely, J., Fein, H. G., . . . Sherman, S. I. (2014). Prognosis of Differentiated Thyroid Cancer in Relation to Serum Thyrotropin and Thyroglobulin Antibody Status at Time of Diagnosis . Thyroid, 35-42. Rego-Iraeta, A., Pérez-Méndez, L. F., Mantinan, B., & Garcia-Mayor, R. V. (2009). Time Trends for Thyroid Cancer in Northwestern Spain: True Rise in the Incidence of Micro and Larger Forms of Papillary Thyroid Carcinoma. Thyroid, 333-340. Savin, S., Cvejić, D., Mijatović, L., & Simonović, S. (2010). Measuring Thyroglobulin Concentrations in Patients with Differentiated Thyroid Carcinoma. Journal of Medical Biochemistry, 243-253. Schlumberger, M. J. (2000). Papillary and Follicular Thyroid Carcinoma. The New England Journal of Medicine , 297-306. Read More