Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241118EnglishN2019April30HealthcareThe Malignant Round Cell Tumors: Histopathological Study and Immunohistochemistry English0107Meghavi R. JoshiEnglish Dhaval JetlyEnglish Mital KundariyaEnglishMalignant round cell tumors include a diverse group of cancers that appear morphologically as round cells. More commonly malignant round cell tumors include cancers consisting of small to intermediate cells having a dark, hyper chromatic nuclei and scant or indistinct cytoplasm. These malignant blue cell tumors include: Primitive neuroectodermal tumor (PNET)/Ewing’s sarcoma family, Neuroblastoma, Non-Hodgkin’s lymphoma, Rhabdomayosarcoma, Wilms tumor, Retinoblastoma, Small cell osteosarcoma, Medulloblastoma, Desmoplastic round cell tumor, Mesenchymal chondrosarcoma and Merkel cell carcinoma. Aims & Objectives: 1. To study Histopathology of Malignant small round cell Tumors. 2. To correlate Histopathological findings with Clinical features, physical findings and imaging studies. 3. To study importance of Immunohistochemistry profiles in Malignant small round cell tumors. Materials & Methods: This is the prospective study and 100 consecutive cases of Malignant round cell tumor received from data of department of pathology during period from 2014 to 2017 were studied. Histological parameters were studied on biopsy fixed in 10% neutral formalin, embedded in paraffin wax and stained with hematoxyline &eosin. IHC stains were performed on each cases. Results: Out of 100 cases, there were 36 cases of Ewing sarcoma/PNET with highest incidence (36%) 21 Cases of Neuroblastoma(21%),15 cases of Non-Hodgkin’s lymphoma (15%). According to age distribution, the highest incidence was observed in 0-14 years of age group. According to sex distribution, highest incidence observed in males. IHC study in Ewing sarcoma /PNET and Neuroblastoma shows positive reaction with CD 99, vimentin and NSE, NB, S100, synaptophysin, chromogranin respectively. IHC study of NHL showing majority are T cell lymphoblastic lymphoma. IHC study of Rhabdomyosarcoma shows positive reaction with desmin, myoD1, Myoglobin, vimentin. Conclusion: Most frequent round cell tumors are Ewing sarcoma /PNET , Neuroblastoma, Non-Hodgkin’s lymphoma, Rhabdomyosarcoma, Wilms tumor, Retinoblastoma. Malignant round cell tumors have male predominance and presented in early childhood. This study highlights the importance of histological examination of resected specimen or biopsied tumors. IHC represents a tool that can provide a clear distinction among the various tumor types. EnglishSmall blue round cell tumors, Ewing sarcoma/PNET, Primitive tumors, Childhood undifferentiated tumorINTRODUCTION Malignant round cell tumors include a diverse group of cancers that appear morphologically as round cells. These may be large undifferentiated malignant neoplasms. More commonly malignant round cell tumors include cancers consisting of small to intermediate cells having a dark, hyper chromatic nuclei and scant or indistinct cytoplasm. These malignant blue cell tumors include: Primitive neuroectodermal tumor (PNET)/Ewing’s sarcoma family, Neuroblastoma, Non-Hodgkin’s lymphoma, Rhabdomayosarcoma , Wilms tumor, Retinoblastoma, Small cell osteosarcoma, Medulloblastoma, Desmoplastic round cell tumor, Hepatoblastoma –[anaplastic form only], Mesenchymal chondrosarcoma, Merkel cell carcinoma. 1 In Malignant round cell tumors, this task is difficult because of similar morphological appearances in histopathology. Through the identification of specific cellular components, using specific monoclonal or polyclonal antibodies, Immunohistochemistry (IHC) has emerged as the most practical adjunct tool to the histopathology making accurate diagnosis possible.9 AIMS & OBJECTIVES To study Histopathology of Malignant small round cell Tumors. To correlate Histopathological findings with Clinical features, physical findings and imaging studies. To study importance of Immunohistochemistry profiles in malignant small round cell tumors. MATERIALS & METHODS Patients diagnosed in our institute as Malignant round cell tumor were selected. This is the prospective study and 100 consecutive cases of Malignant round cell tumor received from data of department of pathology during period from 2014 to 2017 were studied. Clinical finding of patient was obtained by patient’s medical records, relevant clinical history, laboratory investigation and Radiological finding. Histological parameters were studied on biopsy fixed in 10% neutral formalin, embedded in paraffin wax and stained with hematoxyline &eosin .Examine for Architecture and pattern of tumor cells, morphology of cells with nuclear and cytoplasmic characteristics, Presence of mitosis, Hemorrhage, Necrosis or Calcification, any extracellular materials in Stroma, Presence or Absence of Rosettes or Pseudo rosettes. After microscopic examination of routinely stained section followed by special stain [PAS], diagnosis is formulated. IHC stains were performed on biopsy section and IHC was performed on tissue block from each cases using ABC technique with antigen epitope enhancement by heat, the diaminobenzidine reaction was used as final detection step. The slides were counter stained with Mayer’s hematoxylin. The method for epitope retrieval was overnight incubation at 60 degree Celsius. After antigenic epitope enhancement the staining was performed by fully automated machine VENTANA BENCHMARK XT. Appropriate positive and negative controls were included in all stains to ensure quality and consistency of staining results. RESULTS AND OBSERVATION The Malignant small round cell tumors are a heterogeneous group of malignant neoplasms. Most higher incidence noted in our study is Ewing sarcoma /PNET (36%) followed by Neuroblastoma(21%), Non-Hodgkin’s lymphoma (15%) and other small round cell tumor as shows in table no1. These tumors occurs mainly in children up to 14 years of age. Most of the Neuroblastomas &Nephroblastomas (Wilms tumor) appeared in the age group of less than 5 years whereas majority of other Small Round Cell Tumors appeared after the age of 5 years (table 2). Male predominance seen in these tumors. (Table 3). Histopathological features: 36 cases of Ewing sarcoma / PNET  showing Diffuse sheet or lobular pattern of cells, these cells are small, round to oval  with fine to vesicular chromatin with occasional nucleoli  with scanty cytoplasm or some having vacuolated due to glycogen deposition ,with variability in mitosis, necrosis. IHC shows positive reaction as in Table 4. 21 cases of Neuroblastoma showing diffusely arranged small round to oval cell with salt and pepper chromatin with occasional nucleoli, scanty cytoplasm with variable necrosis and rosettes formation and three cases having ganglionic differentiation. IHC shows positive reaction as in Table 5. 9 out of 15 cases of Non-Hodgkin’s lymphoma showing T cell lymphoblastic lymphoma, 3 cases of B cell lymphoblastic lymphoma and 3 cases evaluated as DLBCL by IHC. Table 6. Out of 11 cases of Rhabdomyosarcoma 8 cases shows histological features of Embronal type and 3 cases shows alveolar type Rhabdomyosarcoma. IHC shows positive reaction as in Table 7. 7 Cases of wilms showing microscopically blastemal cells are arranged in nests diffuse sheets, tubules surrounded by mesenchymal cells. These cells are small round with hyper chromatic nuclei, inconspicuous nucleoli with scanty cytoplasm, with variable mitosis and necrosis.IHC shows positive reaction as in Table 8.   All cases of Medulloblastoma showing sheets of small round cells with scanty cytoplasm, hyper chromatic nuclei often elongated or carrot shaped. Mitosis seen. IHC shows positive reaction as in Table 9. There are 2 cases of desmoplastic small round cell tumor shows solid nests of round to oval cells surrounded by cellular desmoplastic stroma. Necrosis is seen. Both cases of DSRCT showing immunoreactivity with AE1, Desmin, vimentin, and NSE. [Figure10, 11] One case of Retinoblastoma showing microscopically sheets and nests of small round cells with scanty cytoplasm, hyper chromatic nuclei. Flexner winter steiner rosettes seen.IHC shows positivity with NSE, Synaptophysin, and S100. One case of pleuropulmonary blastoma microscopically showing cyst wall lining and tumor cells are beneath to lining epithelium. Tumor cells are round to spindled hyper chromatic nuclei. Focal area of necrosis is seen. IHC shows positivity with Vimentin, Desmin, CD117, Ki-67 (high 85%), CD99 (weak) One case of Small cell OS showing diffusely arranged small round to spindly cells, focal area of malignant osteoid seen. IHC shows positivity with Vimentin and S100. Negative for CD99, EMA, LCA, Desmin, Synaptophysin. Discussion Malignant Small round cell tumors comprise a significant fraction of pediatric tumors. Malignant Small Round Cell Tumors is the name given to a group of malignant neoplasms that occur mostly in the pediatric age group (up to 14 Yrs.) These tumors show characteristic appearance under   the microscope, consist of Small Round Cells that Stain Blue on routine Haematoxylin and Eosin stain. 1 Incidence of SBRCT is on rise all over the world 2 Over 60% of all childhood MRCT are now curable.3 In present study out of 100 cases 76 cases are in pediatric age group below 14 years. Distribution of types of tumor in our study compared with other study. Result shows some variation as in Sajid H. Shah5 study 12.5% cases have inconclusive results so not categorized to specific type, however in our study one cases is inconclusive and we also encounter one case of Pleuropulmonary blastoma and two cases of  DSRCT .(Table 10) In our study Most of the Neuroblastomas and Wilms Tumor  appeared in the age group of less than 5 years whereas majority of other malignant round cell tumor appeared after the age of 5 years, similar to Dr. Y. Krishna Bharathi study of 56 cases round cell tumors in pediatric age group 8.(table 11) As shown in our results, ES/PNET is the most common MSRCT. The tumor cells show cytoplasmic immunoreactivity to vimentin and membranous immunoreactivity to CD99. CD45 was negative which ruled out lymphoma. Immunohistochemically, strong membrane staining for CD99 is consistently seen in almost all cases of EWS/PNET, although it is not very specific because it is shown in several other soft tissue sarcomas and lymphoblastic lymphomas. Twenty one case of Neuroblastoma which showed cytoplasmic positivity for Synaptophysin and Chromogranin and NSE. Absence of positive staining for CD99 and CD45 was helpful in differentiating it from EWS/PNET and lymphoma. The markers synaptophysin, and chromogranin and CD56 are restricted to neuroendocrine neoplasms; nevertheless, it should be reemphasized that they are seen in only 30-50% of cases. Neuroblastoma shows immunoreactivity with, Synaptophysin, chromogranin, NSE and CD56.Tumor cells usually do not react with vimentin, CD99, actin or desmin. In present study T-LBL is more compared to those with B-LBL. T-LBL constituting 60% of all lymphoma cases. These cases show younger age, a higher rate of mediastinal mass. IHC was performed in all cases of suspected NHL. The tumor cells show cytoplasmic and membranous immunoreactivity to CD45. Cytokeratin (CK) was negative which excluded poorly differentiated and undifferentiated carcinomas. CD45 is a surface antigen expressed by virtually all hematolymphoid proliferations, and monoclonal antibodies for this marker are reliably specific. We found three cases of Rhabdomyosarcoma which showed cytoplasmic immunoreactivity for vimentin, desmin and CD99, hence subcategorized as alveolar rhabdomyosarcoma. Rhabdomyosarcoma express skeletal muscle markers, like desmin, myoD1, myogenin and CD99 (the alveolar type). Single cases of Retinoblastoma of eye, presented as a big mass involving the eye and extending to the periorbital region. On H&E examination it presented as malignant round cell tumor with rosette formation but for definite diagnosis IHC needed. Four cases of Medulloblastoma were diagnosed in the cerebellum in which tumor cells were so undifferentiated in morphology that making the differentiation from Glioblastoma multiforme (GM), PNET, and metastatic carcinoma very difficult. IHC solved the problem and diagnosis was given in these cases. Two  Abdominal DSRCT cases, One case of  Pleuropulmonary blastoma  and One case of Small cell Osteosarcoma the diagnosis was given by extensive  panel of antibody given on IHC. In only single case IHC is inconclusive even by extensive panel of antibody so given as Undifferentiated Malignant Round Cell Tumor. Immunohistochemistry is playing an increasing role in modern surgical Histopathology.3 The use of extensive panels of antibodies in all Malignant Round Cell  Tumors  allows accurate histological diagnosis in more than 95% cases. Conclusion The MRCTs are a heterogeneous group of malignant neoplasms. It occurs mainly in children up to 14 years of age. Most of the Neuroblastomas &Nephroblastomas (Wilms tumor) appeared in the age group of less than 5 years whereas majority of other Small Round Cell Tumors appeared after the age of 5 years. Male predominance seen in these tumors. Histopathological examination forms the basis for diagnosis of MRCT in the form of features like Small Blue Round Cells with scanty cytoplasm along with specific characteristics like Rosettes, Pseudo rosettes and Neurophils that helps to differentiate the entities of MRCTs. Special stain like PAS is also helpful for diagnosis of ES/PNET family of tumors by positive staining for glycogen. After the Histopathological diagnosis of typing of MRCTs, IHC has to be done in each and every case. IHC represents a tool that can provide a clear distinction among the various tumor types to ensure appropriate and specific treatment. This study highlights the importance of histological examination of resected specimen or biopsied tumors. However, IHC confirms the final diagnosis and also helpful to know the Prognosis of tumor.  In Recent years, role of molecular genetic studies has proved its role as a valuable tool for definitive diagnosis in questionable cases. Acknowledgement: Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors/editors/publishers of all those articles, journals and books from where the literature of article has been reviewed and discussed.     Englishhttp://ijcrr.com/abstract.php?article_id=2594http://ijcrr.com/article_html.php?did=2594 Gregario A, Corrias et.al (July,2008)”Small blue round cell tumors”: Diagnostic& prognostic usefulness of  surface molecule”. Histopathology 53 (1):73-80. doi:10.111/j.1365-2559. 2008. Arya L.S. Childhood cancer-challenges and opportunities. Indian j.of paediatrics.2003;70:159-62. Wick MR. Immunohistochemical approaches to the diagnosis of undifferentiated malignant tumors. Ann Diagn Pathol 2008;12:72-84. Chan JK. Advances in immunohistochemistry: Impact on surgical pathology practice. Semin Diagn Pathol 2000;17:170–7. Sajid hussain shah et al.Frequency of malignant solid tumors in children, JPMA March 2000. Chang TK,LI CY ,Smithson WA. Immunohistochemical study of small round cell tumors in routinely processed specimen.Archpathol lab med 1989;113(12):1343-8. Rossia Antinio G, Nascimentob,Fabio Canala, Angelo Paolo Dei Tosa. Review :Small Round Cell Neoplasm of Soft Tissues: An Integrated diagnostic approach. Current Diagnostic pathology:2007,13150-163 Dr. Y. Krishna Bharathi, Study of pediatric small blue round cell tumors with immunohistochemical correlation IOSR Journal of Dental and Medical Sciences 2279-0853,Volume 15,Issue 6 ,June 2006. Kurtin PJ, Pinkus GS. Leukocyte common antigen-a diagnostic discriminant between hematopoietic and nonhematopoietic neoplasms in paraffin sections using monoclonal antibodies: Correlation with immunologic studies and ultrastructural localization. Hum Pathol 1985;16:353-65.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241118EnglishN2019April30HealthcareFrontal Sinus Variability as a Tool in Forensic Identification- A Pilot Study Using Radiographic Images and Software Analysis English0812Prachi GarhiaEnglish Susmita SaxenaEnglish Anunay GuptaEnglishAim: To evaluate the efficacy of the frontal sinus as an indicator for personal identification Objectives: 1) To determine the presence or absence of symmetry of frontal sinus in each individual and draw comparison between males and females 2) To determine the number of lobes of frontal sinus in each individual and draw comparison between males and females 3) To determine the overall dimensions of frontal sinus in each individual and draw comparison between males and females Materials and Methodology: Paranasal sinus view radiographs of sixty four volunteers visiting the hospital’s outpatient department were randomly selected and the frontal sinuses were analysed using GIMP (GNU Image Manipulation Program) 2.0 software, based on Ribeiro Fde’s measurement criteria. Results: The overall dimensions of the frontal sinus were greater in males as compared to females, with statistically significant difference. Bilateral symmetry of frontal sinus was seen in 55 individuals, while asymmetry was observed in 9 individuals, out of which right dominant asymmetry was found in 6 individuals and 3 volunteers had left dominant frontal sinus. Twenty seven individuals showed less than or equal to four number of lobes, while thirty seven individuals had greater than or equal to five lobes. No cases of unilateral aplasia or complete aplasia were observed. Conclusion: The frontal sinus displayed uniqueness in different individuals and can be used as a valuable aid in terms of personal identification. EnglishForensics, Forensic dentistry, Paranasal sinus viewIntroduction: Forensic dentistry is the sub specialty of dentistry that merges the science of dentistry with the subject of forensics to deal with matters pertaining to legal interest, be it cases of mass disasters or property related disputes. Forensic dentistry initially involved exploiting the uniqueness of the human dentition, dental restorations, and other intraoral details for identification purposes, but is slowly evolving into a more comprehensive and detailed practice. Fingerprint analysis, DNA profiling, anthropological methods, and other techniques can facilitate personal identification.1 However, in cases where the soft tissues of human remains have decomposed, or where DNA is damaged, fingerprint analysis or DNA identification does not seem to be of much use. In such a scenario, anthropological methods are preferred, of which comparative radiography forms a major part.2 Uniqueness of anatomical structures and specific variations in their morphology provide the foundation stone for forensic identification of unknown deceased persons. Among the four paranasal sinuses, namely, maxillary, frontal, ethmoidal, and sphenoidal, frontal sinus has been a topic of significant forensic interest, owing to its unique presentation in each individual.3,4 Frontal sinuses are highly variable in size, ranging from a few cubic centimetres to almost the whole area of the frontal lobe, with an average height of 24.3 mm and an average extent of 29mm from the midline to the lateral wall of the sinus.5 The significance of these sinuses in personal identification has time and again been emphasized by anatomists, radiologists, as well as anthropologists who have stated that no two frontal sinuses are alike.6 Studies have been done to utilize sinus radiography for the identification of remains 7 as well as determination of sex and race.8 The current study was undertaken to study the frontal sinus variations in individuals and to propose its feasibility as a personal indicator in forensic dentistry. Methodology: The current study was carried out in the Department of Oral Pathology and Microbiology, ESIC Dental College and Hospital, New Delhi, after acquiring clearance from the institute’s ethical committee. Paranasal sinus view radiographs of sixty four volunteers visiting the hospital’s outpatient department were randomly selected and evaluated. The sample comprised of thirty eight males and twenty six females within the range of twenty to fifty years, with no history of orthodontic treatment, maxillofacial surgery, trauma, endocrine disturbances, nutritional disorders or any marked facial asymmetry. The radiographs were taken by the same radiologist at 70 kVp, 20 mA, and an exposure time of 3 seconds. The radiographs were then observed and frontal sinuses were analysed by a single trained observer using the GIMP 2.0 software, with the help of radiographic tracings, based on the following criteria: Absence or presence of bilateral symmetry Number of lobulations, based on the indentations present on the sinus outline Greatest height of each frontal sinus Greatest width of each frontal sinus In all the radiographs, the border lines of the frontal sinus were based on Ribeiro Fde’s measurement criteria (Fig. 1). 9 The reference lines and measurements used are as follows: Reference base line : a line passing through the superior border of orbit (A) Distance between the upper limit of the orbits and the highest point of the right frontal sinus (B) Distance between the upper limit of the orbits and the highest point of the left frontal sinus (C) Line marking the maximum lateral limit of the right frontal sinus (D) Distance between the lateral most boundary of the right frontal sinus and the line passing upwards along the nasal septum (E) Line marking the maximum lateral limit of the left frontal sinus (F) Distance between the lateral most boundary of the left frontal sinus and the line passing upwards along the nasal septum (G) Results: The data was compiled using Microsoft Excel Spreadsheet and subjected to statistical analysis using Statistical Package of Social Sciences (SPSS).Version 20. Shapiro Wilk’s test was done to determined normality of the data. Data was found to be normally distributed, thus parametric tests were done. Unpaired T-test was done to compare the height and width of the sinus among male and female groups. Chi square test was used to find out if there was any statistically significant difference in symmetry and number of lobes in the sinus of males and females. The measurements of the study reveal that the overall dimensions of the frontal sinus were greater in males as compared to females. On applying the unpaired t test for comparing the width and height of the sinuses in both the genders, the results were statistically significant (P value < 0.05). (Table 1) Bilateral symmetry of frontal sinus was seen in 55 individuals, i.e., 31 males and 24 females, while asymmetry was observed in 9 individuals (7 males and 2 females), out of which right dominant asymmetry was found in 6 individuals and 3 volunteers had left dominant frontal sinus. Chi square test was applied on the aforementioned data and the result between both the genders was not statistically significant (P value > 0.05). (Table 2) Twenty seven individuals showed less than or equal to four number of lobes, while 37 individuals had greater than or equal to five lobes. The results were statistically insignificant when Chi square test was applied to compare the number of lobes as recorded for both the genders. (P value > 0.05) (Table 3) No cases of unilateral aplasia or complete aplasia were seen in the population under study. Discussion: The application of X-ray in the field of forensics was introduced by Professor Arthur Schuster in 1896, for demonstration of the presence of bullets inside a victim’s head.10 Schüller was the first person to propose the utilization of the radiographic images of the frontal sinuses for the purpose of identification, while Culbert and Law were the first to describe the complete process of radiological identification using the pneumatic spaces found within the skull. 11,12,13 The uniqueness of the anatomy of the frontal sinus makes it a helpful aid in forensic dentistry. These sinuses are considered similar to fingerprints, owing to the fact that each frontal sinus is distinctive and unique.2 There are various factors that make the frontal sinus a reliable indicator for personal identification: It is a highly variable anatomical structure, with even the frontal sinuses of monozygotic twins showing considerable difference 8 It is considered to be relatively stable during the adult life until old age, when pneumatisation may occur owing to atrophic changes 14 It has strong surrounding walls and is found to be intact in human remains. Being posterior to the thick outer table of frontal bone further enhances its stability.15 It has been reported that approximately 800-1600 foot pounds of force is required to fracture the walls of the frontal sinus, as can be seen in victims of high impact accidents or gunshot injuries.16,17 The morphology of the frontal sinuses remains practically unchanged during one’s entire adult life, although some environmental factors can modify its structure, such as hyperpneumatization from sports activities, illnesses, infections, tumours or trauma, among others. It has been reported that the frontal sinuses are smaller and more scalloped in females.18,19The results of the present study revealed that the dimensions of the frontal sinus are greater in males as compared to females, which seems to agree with the results of the studies carried out by Chaudhary and Singh, and Ponde et al.1,18 Symmetry was seen in 85.9% of the cases under study, while the percentage of people with bilateral frontal sinus symmetry was comparatively lower in the study conducted by Chaudhary and Singh1, and David and Saxena10, with 62% and 58% of the individuals showing symmetry respectively. The results were significantly lower for the study conducted by Tang et al on Japanese population with only 43.1% showing bilateral symmetry. [19] Asymmetry was observed in 14.1% of the patients, while it was reported to be 30% and 56.6% in the studies conducted by Chaudhary and Singh, and Tang et al respectively.1,19 No cases of sinus aplasia were observed in the present study, as opposed to Kullman who reported frontal sinus absence in 5% of the cases.20 The method used for frontal sinus analysis is quite easy and can be easily employed by a general dentist as well. Paranasal sinus view radiographs are quite commonly recorded for diagnostic purposes and require equipment that are generally available in a hospital set up, thus making its application quite feasible. Certain limitations related to the use of frontal sinus a tool for personal identification do exist. Frontal sinus has been reported to be affected by environmental as well as genetic factors. Growth hormone or somatotropin has also been stated to affect the sinus morphology.[6] Errors may also occur due to slight differences in the radiographic technique used by different operators, which may be overcome by restricting the use to a single operator. Conclusion: The study undertaken revealed that the frontal sinuses of the sixty four volunteers did not match and it was concluded that frontal sinus displayed uniqueness in different individuals and can be used as a valuable aid in terms of personal identification. The method used was very simple and can be easily applied by any general dental practitioner. The radiograph required for the study, i.e., paranasal sinus view, can be easily obtained. However, establishing the authenticity of the frontal sinus as a personal indicator requires further studies on larger samples, with the implementation of other parameters such as age. Acknowledgements: Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. The authors would also like to thank Dr. Roomani Srivastava, Tutor, Department of Public Health Dentistry, ESIC Dental College and Hospital, Rohini, New Delhi, for her inputs on statistical analysis. Ethical clearance: Granted Patient consent: Taken Source of funding: None Conflict of interest: None Englishhttp://ijcrr.com/abstract.php?article_id=2595http://ijcrr.com/article_html.php?did=2595[1]Chaudhary S., Singh U., Uniqueness of Frontal Sinus as a Personal Identification in Forensic Odontology. Ann. Int. Med. Den. Res. 2016; 2(5):AT07-AT09 [1].Divakar KP., Forensic Odontology: The New Dimension in Dental Analysis. Int J Biomed Sci. 2017;13(1):1–5. [1].Harris AM, Wood RE, Nortjé CJ, Thomas CJ. The frontal sinus: forensic fingerprint? A pilot study. The Journal of forensic odonto-stomatology. 1987 Jun;5(1):9-15. [1]. Harris AM, Wood RE, Nortjé CJ, Thomas CJ. Gender and ethnic differences of the radiographic image of the frontal region. The Journal of forensic odonto-stomatology. 1987 Dec;5(2):51-7. [1]. Donald PJ, Gluckmann JL, Rice DH. The Sinuses. New York: Raven Press; 1994. p.41-44 [1]. Nambiar P, Naidu MD, Subramaniam K., Anatomical variability of the frontal sinuses and their application in forensic identification. Clinical Anatomy: The Official Journal of the American Association of Clinical Anatomists and the British Association of Clinical Anatomists. 1999;12(1):16-9 [1]. Kanchan T, Krishan K, Menezes RG, Suresh BK, Lobo SW. Frontal sinus radiographs--a useful means of identification. Journal of Forensic and Legal Medicine. 2010 May;17(4):223-4. [1]. Igbigbi PS, Nanono-igbigbi AM. Determination of sex and race from the subpubic angle in Ugandan subjects. The American journal of forensic medicine and pathology. 2003 Jun 1;24(2):168-72. [1].Ribeiro Q, de Andrade F. Standardized measurements of radiographic films of the frontal sinuses: An aid to ENT: Ear, Nose & Throat Journal. 2000 Jan 1;79(1). [1]. Eckert, W.G. & Garland N. History of the forensic applications in radiology. The American Journal of Forensic Medicine and Pathology.(1984). Vol. 5, No. 1, p. 53–56, ISSN:0195-7910 [1].CULBERT WL, LAW FM. Identification by comparison of roentgenograms: of nasal accessory sinuses and mastoid processes. Journal of the American Medical Association. 1927 May 21;88(21):1634-6. [1].Gruber J., Kameyama MM., The role of radiology in forensic dentistry. Pesquisa Odontológica Brasileira. 2001 Sep;15(3):263-8 [1]. Carvalho SPM, Silva RHA, Lopes Jr C, Sales-Peres A., Use of images for human identification in forensic dentistry. Radiol Bras. 2009;42(2):125–130. [1].Yoshino  M, Miyasaka S, Sato H, Seta S. Classification system of frontal sinus patterns by radiography.Its application to identification of unknown skeletal remains. Forensic  Science International. 1987 Aug 1;34(4):289-99. [1]. Marlin DC, Clark MA, Standish SM. Identification of human remains by comparison of frontal sinus radiographs: a series of four cases. Journal of Forensic Science. 1991 Nov 1;36(6):1765-72. [1].Nahum AM. The biomechanics of maxillofacial trauma. Clinics in plastic surgery. 1975 Jan;2(1):59-64. [1].Wallis A, Donald PJ., Frontal sinus fractures: a review of 72 cases. The Laryngoscope. 1988 Jun;98(6):593-8. 18.Schuller A. A note on the identification of skulls by X-ray pictures of the frontal sinuses. Med J Aust. 1943 Jun 19;1:554-6. 19. Tang JP, Hu DY, Jiang FH, Yu XJ. Assessing forensic applications of the frontal sinus in a Chinese Han population. Forensic Science International. 2009 Jan 10;183(1-3):104-e1. 20. Kullman L, Eklund B, Grundin R. Value of the frontal sinus in identification of unknown persons. The Journal of forensic odonto-stomatology. 1990 Jun;8(1):3-10.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241118EnglishN2019April30HealthcareIn vivo study of Depigmentation Using Tyrosine Ammonia Lyase from Trigonella Foenum-graecum on Zebra Fish Embryos English1316Kavitha G. Singh; Umme Umaima S.; Khushbu JangidEnglishAim: To extract Tyrosine ammonia Lyase(TAL) from Trigonella foenum-graecum and utilize it for the treatment of depigmentation by carrying out in-vivo analyses on Zebra fish embryos Tyrosine ammonia Lyase is an enzyme that is involved in deamination of L-tyrosine to P-coumaric acid. The enzyme TAL was extracted from Trigonella foenum-graecum. This enzyme has been targeted to be used in depigmentation of the skin in the current study using zebra fish as a model organism. This enzyme participates in depigmentation by inhibiting the production of melanin pigment, where in conversion of L-tyrosine in the melamine synthesizing pathway has been prevented by deaminating L-tyrosine to P-coumaric acid. The methodology of separation of proteins using acetone has been implemented in the extraction of the enzyme. Standardization of enzyme was accomplished to detect the presence of the enzyme. The formation of P-coumaric acid indicated the presence of the enzyme that was detected utilizing UV-Visible spectrophotometer at 380nm. In-vivo experimental study was carried on the zebra fish embryos for detecting the presence of the enzyme that was confirmed by observing the depigmented patterns in the developed embryos that were been treated with TAL. EnglishL-tyrosine ammonia Lyase, Depigmentation, P-coumaric acid, Trigonella foenum-graceum, Zebra fishINTRODUCTION Depigmentation is a process where the synthesis of melanin pigment is inhibited resulting in de-coloration of skin. The melanocytes losses their ability to synthesize melanin pigment by inhibiting the further conversion of L-tyrosine to participate in the melanin synthesizing pathway, by deaminating L-tyrosine to P-coumaric acid. The purpose of the current study is to detect the presence of the enzyme tyrosine ammonia Lyase by extracting it from the leaves of Trigonella foenum-graecum, and there by utilizing it to cause depigmentation. The color of the body is induced by melanin pigment that is produced by melanocytes which are present in the lower layer of the epidermis. Tyrosine ammonia Lyase is an enzyme that deaminates L-tyrosine to P-coumaric acid, as a result of which the conversion of L-tyrosine  to further products in the pathway is eliminated and the melanin pigment from the melanocytes is not synthesized. Trigonella foenum-graecum also known as fenugreek plant, belong to the family of Fabaceae, Papillionaceae subfamily. It is an efficient aromatic herb found mostly in Asia and Europe and also in North African areas. It is an ancient plant that is used in treatment of various types of diseases, among which diabetes mellitus is one of them. The leaves of the plant are used as spice in many dishes to improve the taste; the leaves are also known as methi leaves.   Zebra fish is a fresh water  living organism  found usually in  Himalayas and some parts of west Bengal. It belongs to minnow family, It is the most frequently used model organism in most of the research work carried out through conducting in-vivo experiments. The zebra fish genome is fully sequenced and shows 80% of genomic similarities with that of human genomic sequence and hence has been considered to be most frequently used model organism all over the globe to carry out medicinal research work. The best examples of it are conditions like Parkinson’s disorder, alziemer, and atherosclerosis. The embryonic developmental stages have been well studied and it rapidly develops. The embryos occur to be transparent, with natural pigmentation. It also has the capability to undergo external fertilization. MATERIALS Reagents: Chilled acetone, Chilled distilled water, Tris HCL, HCL (1N), NaOH (1N), L – tyrosine, Methanol. Instruments: Chilled grinder, Cooling centrifuge, UV spectrophotometer, Computer, Incubator, Refrigerator,  pH Meter. Other Requirements: Glass wares, Mortar & pestle, Centrifuge tubes, Muslin cloth, Fresh leaves of Trigonella foenum-graecum. METHODOLOGY Preparation of Tyrosine Ammonia Lyase: Trigonella foenum-graecum leaves were collected from north areas of Bangalore. Leaves were washed and air dried to remove water content. 26.6g of leaves were weighed and grinded with 100ml of distilled water using a mixer grinder. The prepared mixture was filtered using muslin cloth. The filtrate was measured; its volume was found to be 110ml that was refrigerated in freezer for 24 hours. Then to the filtrate equal amount of chilled acetone was added and kept undisturbed in freezer at -20 degree C for 24 hours. After precipitation two layers were formed, upper layer were discarded and the precipitate containing the protein was collected. Then the precipitate was centrifuged at 5000 rpm for 5 minutes at low temperature in cooling centrifuge. The pallet was collected, washed using acetone and the mixture was then poured into Petri plate for acetone to dry. The acetone powder obtained was weighed. 1.0g of the powder was weighed and homogenized with 20ml of 1mM Tris HCl (pH=8.2) utilizing a pestle and mortar. The mixture obtained was centrifuged at 5000 rpm for 5 minutes. The crude enzyme extract was obtained in a supernatant form which was used for further analysis. (Umme Umaima et al., 2016) (Sarina P. Khabade, 2014) Enzyme Assay of Tyrosine Ammonia Lyase: Reaction mixture tube was prepared with 1ml of enzyme source, 0.2ml of 1mM L-Tyrosine (substrate) and 0.8 ml of 0.1M Tris HCl buffer (pH=8.9). A control tube was also prepared using 1ml of distilled water instead of 1ml of enzyme extract. Both the tubes were incubated in incubator at 37°C for 30 minutes. After incubation reaction is terminated by the addition of 0.5 ml of 1N HCl. Tyrosine Ammonium Lyase enzyme deaminates L-Tyrosine and produce p-coumaric acid with the release of ammonia. Then it was quantitatively measured by using UV – Visible spectrophotometer at 380nm. (Umme Umaima et al., 2016) Experiment on Zebra Fish (In-Vivo) To reach depigmentation: Zebra fishes were maintained in lab condition and allowed to mate in a breeding tank. Embryos of Zebra fish were collected  after 20 hours of fertilization before the pigmentation was produced and were transferred in a Petri plate containing embryo water. Two Petri plates containing zebra fish embryos were taken and marked as control and test. Control plate with the zebra fish embryos was left as such without the addition of  enzyme Tyrosine ammonia Lyase, where as test Petri plate with zebra fish embryos was treated with  1 ml of enzyme extract of Tyrosine Ammonia Lyase. Both the plates containing zebra fish embryos were kept at optimum temperature and observed under microscope after 24 hours of incubation. After observation the embryo water in the petri plate was cleaned and the plates were again added with enzyme Tyrosine Ammonia Lyase. Then the petri plates containing zebra fish embryos were further incubated at optimal temperature and were observed after 24 hours for better result. (Umme Umaima et al., 2016) (Sarina P. Khabade, 2014) RESULT Presence of enzyme Tyrosine Ammonia Lyase was confirmed in Trigonella foenum-graecum. Enzyme was assayed as per the standard conditions. UV-Visible spectrophotometer was used to check the activity of enzyme at 380nm. When the in-vivo experiment was performed on the zebra fish embryos, showed a gradual decrease in the pigmentation of the test samples induced with particular concentration of enzyme when compare to the control. Discussion The test sample (Trigonella foenum-graecum) in comparison   with that of control indicated  the presence of  Enzyme tyrosine Ammonia Lyase  in a rich amount as a result of conversion of L-Tyrosine to p-coumaric acid by the action of enzyme(TAL). The presence of enzyme in a higher concentration   in the sample   enables the use of the sample  to carry out the  depigmentation  experiment through carrying out  in-vivo study on zebra fish embryos. The clear indication of depigmentation is obtained through observing the above figure 2 and 3.        The  above  figure2 and  figure3 that represent the embryos in the control sample ( figure 2) that is left untreated  with  enzyme Tyrosine Ammonia Lyase and  the embryos in the test sample ( figure 3) treated with the enzyme  Tyrosine Ammonia Lyase. The absence of   pigmented stripes in the test sample embryos  (figure 3)  indicates the identical effect  of the enzyme obtained through the sample Trigonellafoenum- graecum  which resulted in Depigmentation. Conclusion                          Leaves of Trigonella foenum-graecum are found to contain numerous useful and multi nutritive values, hence is used as herb in many Ayurveda medicines in case of treatment of many diseases, one of them is diabetes mellitus. Depigmentation is characterized as condition where in skin becomes white, as result of inhibition of melanin pigment that is responsible for darkening of skin color. There are many therapeutics and sunscreens creams being used in obtaining the depigmented state, which are useful only for a short period, moreover there are many side effects that get induced affecting the skin and sometimes other parts of the body. As a result of this consequences Trigonella foenum-graecum containing multi nutritive value, medicinal properties along with its zero side effect inducing ability, has been implemented to be utilized inducing depigmentation in the current study and as well as can be utilized in the treatment of many other diseased condition. Englishhttp://ijcrr.com/abstract.php?article_id=2596http://ijcrr.com/article_html.php?did=25961.  P Khosla, DD Gupta, RK Nagpal.  Effect of Trigonella foenum graecum ( Fenugreek) on blood giucose normal and diabetic rats. Indian journal of physiology and pharmacology 39, 173-173, 1995. 2.   S Kaviarasan, GH Naik, R Gangabhagirathi, CV Anuradha, KI Priyadarsini.  In vitro studies on antiradical        and antioxidant activities of fenugreek (Trigonella foenum graecum) seeds. Food chemistry 103(1), 31-37, 2007. 3.   K Srinivasan.  A review of Health Beneficial Physiological  Effect: Fenugreek(Trigonella foenum graceum). Food review international 22(2), 203-224, 2006. 4.   Zia Tayyaba, S Nazrul Hasnain,  SK Hassan. Evaluation of oral hypoglycemic effect of Trigonella Foenum graceum(Fenugreek) in normal mice. Journal of ethnopharmacology 75(2-3), 191-195, 2001. 5.  Kowichi Jimbow, Hiroko Obata, Madhu A Pathak, Thomas B Fitzpatrick. Mechanism of De-pigmentation by hydroquinone. Journal of investigation Dermatology 62(4), 436-449, 1974           6.  Gail K Naughton, Diana Reggiardo, Jean-Claude Bystryn. Correlation between vitiligo antibodies and extent of  De- pigmentation in vitiligo. Journal of American Academy of Dermatology 15(5), 978-981, 1986 7.  TP Dooley. Topical skin  de- pigmentation agents: current products and discovery of novel  inhibitors of  melanogenesis, Journal of Dermatological Treatment 8(4), 275-283, 1997. 8.  K M AlGhamdi, A Kumar. De-pigmentation therapies for normal skin in vitiligo universalis. Journal of the European Academy of Dermatology and Venereology  25(7), 749-757, 2011. 9.  Jens Rosler, Florian  Krekel, Nikolaus Amrhein, Jurg Schmid.  Maize phenylalanine ammonia-lyase has tyrosine ammonia- lyase  activity . Plant physiology 113(1), 175-179, 1997. 10.  M Jason MacDonald, Godwin B D’Cunha. A modern view of  phenylalanine ammonia lyase. Biochemistry and Cell Biology 85(3), 273-282,  2007. 11. Robert Gerlai. Zebra fish: an uncharted behavior genetic model. Behaviour genetics 33(5), 461-468, 2003. 12.  Charles B Kimmel, Jill  Patterson, Richard O Kimmel. The development and behavioral Characteristics of the startle response of  Zebra fish. Developmental psychobiology 7(1),  47-60, 1974. 13.   Charles B Kimmel, William W Ballard, Seth R Kimmel, Bonnie Ullmann, Thomas F Schilling. Stages of embryonic development of the  Zebra fish. Developmental dynamics 20393), 253-310, 1995. 14.   John H Postlethwait, Yi-Lin Yan, Michael Gates, Sally Horne, Angel Amores, Alison. Vertebrates Genome evolution and the zebra fish gene map. Nature genetics 18(4), 345, 1998.  15.  Graham J Lieschke, Peter D Currie. Animals models of human disease: zebra fish swim into view, Nature Reviews Genetics 8(5), 353,  2007. 16.  LK Cole, LS Ross. Apoptosis in the developing zebra fish  embryo. Developmental  biology 240(1), 123-142, 2001.