Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411015EnglishN2018August16HealthcarePlatelet Rich Fibrin & Guided Tissue Regeneration Aided Coverage of a Mucosal Fenestration – An Interdisciplinary Approach   English0105Divya S.English Deepika P. C.English AmbikathanayaEnglishAim: Mucosal fenestrations affecting permanent teeth are clinically challenging because they require a more complex approach. The objective of this case report was to describe a treatment modality used to manage an apical fenestration placed on the left mandibular central incisor root. Case Report: The case report describes the management of a patient with mucosal fenestration of root apex. Mucosal fenestration of root apex was treated by a combination of root canal treatment and surgery. Root-end resection was performed to bring the root apex within the alveolus before root-end filling and packing of the bony defects with platelet rich fibrin. The dehiscence of the buccal labial plate was managed by placement of a barrier membrane. The edges of the soft tissue defect was then deepithelialized, approximated and sutured. Discussion: Various treatment modalities advocated in the literature for the management of mucosal fenestration include- root canal treatment and root-end resection, blind root surface instrumentation and mouth rinsing with chlorhexidine, full thickness mucogingival flap with primary or secondary healing, pedicle flap operations, epithelialized and non-epithelialized grafting procedures for root coverage and full thickness mucogingival flaps with guided tissue regeneration and bone grafting. Conclusion: The endodontic and periodontal surgical techniques used in the management of alveolar or mucosal fenestrations alone are unremarkable but combining them can give optimum outcome.  EnglishPlatelet rich fibrin, Mucosal fenestration, GTINTRODUCTION: The relationship between the alveolar bone and the teeth have been dated back to 1963, when O’Connor studied the relationship of teeth with the tooth anatomy, inter-proximal bone, bony wedges and the presence of fenestrations.1 Fenestrations and dehiscences, are being more considered normal variations with regard to presence of the teeth, than pathologic conditions. The criteria for their identification as put forth by Davies RM et al 2. as dehiscence is a lack of cortical bone at the level of the root of the tooth, at least 4 mm apical to the margin of the inter-proximal bone; whereas fenestration is a contained defect of the alveolar bone that exposes the apical or the middle third of the root surface, without involving the marginal bone. A precise and seldom encountered spectacle is a combination of apical fenestration along with a mucosal fenestration. Mucosal or apical fenestration is termed as a pathologic condition which is characterized by the perforation of the cortical plate and the overlying mucosa by the prominent roots of teeth. It was first described in literature as ‘‘bone fenestration by roots of deciduous teeth’’ by Mene´ndezin 19673. Mucosal fenestration may be attributed to decreased thickness of the alveolar housing, labioversion of the tooth in the dental arch, contour of the root apex, occlusal factors, orthodontic tooth movement, periodontal and endodontic pathology and aberrant frenal attachment.4,5,6 Mucosal fenestration is most frequently found in the mandibular or maxillary anterior teeth region, particularly on the labial aspect because of tooth angulation that places the root apices in alabial version. Mucosal fenestrations have been reported in literature but are far less prevalent as compared to normal fenestration perhaps due to symptom free nature.7Even though they are usually symptom-free, they might act as plaque-retaining areas, resulting in irritation and inflammation of the surrounding mucosa.8 Mucosal fenestrations affecting permanent teeth are clinically challenging because they require a more complex approach. The aim of this case report was to represent a treatment modality used to manage an apical fenestration occured on root surface of the left mandibular central incisor. CASE REPORT: A 26 year old male patient reported to the OPD of JSS Dental College and Hospital, Mysore with discoloured lower front tooth. Patient gave a history of trauma 1 year back, following which there was a progressive discoloration of the tooth with intermittent pus discharge. Patient was in good health. No pathologic signs were evident through facial inspection. Oral examination revealed the apical third of the root of 31 that perforated the buccal cortical plate and adjacent alveolar mucosa, being therefore exposed to the oral environment and the discoloured crown of the same tooth. [Fig:1] There was presence of plaque, calculus and soft debris on the exposed root tip and the margins of the surrounding mucosa were inflammed and tender. The tooth was grade 1 mobile. Pretreatment radiograph revealed discontinuity in the lamina dura around the apex of the 31 with periapical radioluscency [Fig:2]. Also, the tooth was nonvital. The probing depth measured 2mm buccally and 1.5 mm lingually hence, diagnosis of asymptomatic chronic apical periodontitis with mucosal fenenstration on tooth 31 was made. TREATMENT: After phase 1 therapy consisting of scaling and root planing, multivisit endodontic treatment was performed. Following working length determination, biomechanical preparation was done using K-file and recapitulation with 5.25% sodium hypochlorite.calcim hydroxide paste was used as intracanal medicament for 10 days. Next appointment, calcium hydroxide paste was removed and root canal was obturated with cold lateral condensation of gutta percha with zinc oxide ugenol as sealant. Surgery was undertaken 2 weeks after endodontic treatment. Fibre reinforced composite splinting was done in relation to 41,31 and 32.  Region 31,32,41,42 was anaesthetized with 2% lignocaine. Crevicular and vertical releasing incisions were made, and full-thickness flap was raised, revealing dehiscence of the buccal cortical bone along the root surface of 31 [Fig:3] Granulation tissue was immediately apparent surrounding the root apex. Curettage of this tissue revealed the extent of bone loss. The prominent portion of the affected root apex was resected. The resection was carried up to the level of remaining sound alveolar bone, so that the root margins were flush with the surrounding bone to reposition the affected root in the alveolar housing [Fig. 4a,b]. Any remaining granulation tissue was removed. The root planning was done and then glass ionomer cement(GC Fuji II; GC America, Alsip, IL)was placed over the prepared root end.[Fig: 6] Autologous graft (platelet rich fibrin) was obtained from the patient and placed in the defect at the periapical region of 31. The root was then covered with GTR membrane (Healiguide).[Fig:6,7] The edge of the mucosal defect was de-epithelialized, approximated and sutured with resorbable sutures to ensure closure [Fig:8]. The wound was covered with periodontal dressing (coe-pak) Post-operative instructions were given and antibiotics and analgesics were prescribed. Patient was recalled after 1 week for removal of the pack. Patient was without any local complications and free of symptoms.  The 2 and 5 months recall showed excellent soft tissue healing [Fig:9a ,b] Periapical radiograph revealed well performed endodontic treatment, healing and remodelling of the apical area at 6 months recall [Fig:10]. A two year follow-up period with every 6 months radiographic evaluation was recommended. DISCUSSION: Mucosal fenestration is a relatively uncommon complication of pulpo-peri radicular disease.9,10,11 The exact aetiology is unknown but review of literature suggests that abnormally labioversed root tips, very thin labial plates and the presence of chronic periapical inflammation may be the probable cause. The first step toward management of mucosal fenestrations is identifying the precipitating cause of its occurrence. In this case, trauma could be the primary etiologic factor, along with prominent root positions and chronic periapical inflammation that might have destroyed the overlying thin facial cortex and mucosa. Mucosal breakdown and exposure of the root tip to the oral cavity leaves the root-tip vulnerable to plaque accumulation and calculus formation which prevents the reformation of mucosal covering. Various treatment modalities advocated in the literature for the management of mucosal fenestration include- root canal treatment and root-end resection,8,9 blind root surface instrumentation and mouth rinsing with chlorhexidine10, full thickness mucogingival flap with primary11 or secondary healing12, pedicle flap operations4, epithelialized and non-epithelialized grafting procedures for root coverage and full thickness mucogingival flaps with guided tissue regeneration and bone grafting13. In this case, since the tooth was non vital, it was first treated by root canal therapy to eliminate the focus of infection. Healing of the mucosa was then aided by removal of the granulation tissue, root end resection, and removal of adjacent infected cementum by root planning. The main objective of resection of the abnormally prominent root apices about the level of remaining sound alveolar bone is to return the root within the bony limit of the cortex. This affords a favourable anatomic configuration and eliminates future risk of irritation. Glass ionomer cement was used to achieve a good apical seal after proper isolation. Jhaveri et al14 have reported light cure GIC to be a successful root end filling material. In the present case, after flap elevation dehiscence of the labial cortical plate was seen. Hence an attempt to regenerate the periodontal supporting tissues was made utilizing the principles of guided tissue regeneration. GTR was used in our case not only as a root coverage procedure, but also as a strategy of endodontic surgery where the ultimate goal was to regenerate the attachment apparatus. Scientific evidence indicates that principles of GTR can be successfully applied in endodontic surgery to correct alveolar bone defects confined to periapical region. Animal histological studies showed that complete bone filling of periapical bone cavities occurred after endodontic surgery only when a barrier for guided tissue regeneration was used, whereas extensive connective tissue filling of the defects was found after conventional endodontic surgery. The present case is unique in that it is the first case to utilize platelet rich fibrin (PRF) as a grafting material. The three crucial factors for healing and soft tissue maturation are angiogenesis, growth factors and mesenchymal stem cell activity. PRF consists of a fibrin 3D mesh polymerized in a specific structure, incorporating platelets, leucocytes, growth factors and circulating stem cells.17 The concentrations of growth factors within the platelet concentrate up regulate cellular activity and subsequently promote periodontal regeneration in vivo. Fibrin, fibronectin, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and transforming growth factor beta (TGF-β)  in PRF aid in angiogenesis, tissue repair and regeneration.18 Hence owing to these advantages PRF was used as an effective biomaterial as projected in this case. CONCLUSION: The endodontic and periodontal surgical techniques used in the management of alveolar or mucosal fenestrations alone are unremarkable but combining them can give optimum outcome. Guided tissue regeneration in combination with platelet rich fibrin can successfully be used to treat fenestrated root apices. Abbreviations: GTR: guided tissue regeneration GIC: glass ionomer cement PRF: platelet rich fibrin PDGF: platelet derived growth factor VEGF: vascular endothelial growth factor TGF-β: transforming growth factor beta 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 for this article has been reviewed and discussed. Conflict of interest: Nil Englishhttp://ijcrr.com/abstract.php?article_id=2509http://ijcrr.com/article_html.php?did=2509 O’Connor TW, Alveolar bony contours, A thesis submitted to the Faculty of Baylor University Dallas, Texas, 1963. Davies RM, Downer MC, Hull PS, Lennon MA, Alveolar defects in human skulls, J Clin Periodontol, 1974, 1(2):107–111. Mene´ndez OR. Bone fenestration by roots of deciduous teeth. Oral Surg Oral Med Oral Pathol 1967;24:654–8. Ju YR, Tsai HY, Wu YJ. Surgical intervention of mucosal fenestration in a maxillary premolar: a case report. Quintessence Int. 2004;35:125e128. Abdelmalek RG, Bissada NF. Incidence and prevalence of alveolar bone dehiscence and fenestration in dry human Egyptian jaws. J Periodontol 1973;44:586–8. Edel A. Alveolar bone fenestrations and dehiscences in dry Bedouin jaws. J Clin Periodontol 1981;8:491–9. Ling LJ. The treatment of fenestrated root: case reports. J Dent Sci. 1989;9:137e140 Dawes WL, Barnes IE. The surgical treatment of fenestrated buccal roots of an upper molar: a case report. Int Endod J 1983;16:82–6. Lehman J III, Meister F Jr, Gerstein H. Use of a pedicle flap to correct an endodontic problem: a case report. Journal of Endodontics 1979;5, 317–20. Lin LJ. The treatment of fenestrated root: case reports. Journal Dental Science1989; 9, 137–40. Tseng C-C, Chen Y-HM, Huang C-C, Bowers GM Correction of a large periradicular lesion and mucosal defect using combined endodontic and periodontal therapy: a case report. International Journal of Periodontics and Restorative Dentistry 1995;15, 377–83. Rawlinson A. Treatment of a labial fenestration of a lower incisor tooth apex. British Dental Journal1984; 156, 448–9. Chen G, Fang CT, Tong C. The management of mucosal fenestration: a report of two cases. Int Endod J. 2009;42(2):156e164. Jhaveri HM, Amberkar S, Galav L, Deshmukh VL, Aggarwal S, Management of mucosal fenestrations by interdisciplinary approach: a report of three cases, J Endod, 2010, 36(1):164–168. Von Arx T, Cochran DL, Rationale for the application of the GTR principle using a barrier membrane in endodontic surgery: a proposal of classification and literature review, Int J Periodontics Restorative Dent, 2001, 21(2):127–139. Pecora G, De Leonardis D, Ibrahim N, Bovi M, Cornelini R, The use of calcium sulphate in the surgical treatment of a ‘through and through’ peri radicular lesion, Int Endod J, 2001,34(3):189–197. Choukron J, Diss A, Simonpieri A et al. Platelet rich fibrin. A second generation platelet concentrate. Part IV. Clinical effects on tissue healing. Oral Surg Oral Med Oral Pathol Oral Rad Endod 2006; 101: e 56-e60. Dvorak HF, Harvey VS, Estrella P, Brown LF, McDenagh J, Dvorak AF. Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Lab Invest 1987;  57: 673-86.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411015EnglishN2018August16HealthcareLeiomyosarcoma of Superior Vena Cava and Inferior Vena Cava - Two Rare Case Reports   English0609Puja KhannaEnglish Shilajit Bhattacharya EnglishObjective: This article attempts to describe two cases of vascular leiomyosarcoma, one arising from the superior vena cava in a 22 year old male patient, and the other arising from the inferior vena cava in a 53 year old female. The objective was to investigate and diagnose the two cases. Material and Methods: Imaging studies (CT scan) were done in both the cases subsequent to initial clinical examination. After surgical resection, the specimens of the tumors were sent to the Pathology Department for histopathological examination. The sections were stained using Haematoxylin and Eosin (H and E). Immunohistochemistry (IHC) was also done. Results: On gross examination, the first case showed a segment of a large vessel with a mass attached to the inner wall and protruding outside through the lumen. Tumor on cut section was solid, firm, homogenous grayish white with focal necrotic areas. In the second case, the vessel wall with an attached firm to hard lesion was received. On histological examination, both the cases were diagnosed to be malignant spindle cell tumor. Immunohistochemical staining revealed that the tumor cells were diffusely positive for SMA and Desmin and negative for S100 and CK. The two cases were reported as vascular leiomyosarcoma, one of the Superior vena cava and the other case of Inferior vena cava. Conclusion: Vascular leiomyosarcoma is a very rare tumor and the Inferior vena cava is the most common site (50%). The Superior vena cava (SVC) is exceptionally involved and only a dozen cases have been previously reported. An accurate imaging and histopathological diagnosis is essential as they can affect the prognosis and treatment approach. So such cases should be thoroughly examined and followed.  English Inferior vena cava Superior vena cava, Vascular leiomyosarcomaINTRODUCTION Vascular leiomyosarcoma is a very rare tumor and the inferior vena cava is the most common site (50%).1 The superior vena cava (SVC) is exceptionally involved and only a dozen cases have been previously reported.2 Leiomyosarcoma is considered a very locally aggressive tumor with rare distant dissemination and an unfavorable prognosis. An aggressive therapeutic approach, including chemoradiotherapy and large surgical resection, is usually considered the treatment of choice.3 CASE REPORT The first case was a 22 year old male who came to the medical outpatient department with history of facial edema, engorgement of neck veins, hoarseness of voice and productive cough since 2 months. CT scan of thorax revealed an intraluminal enhancing lesion in the distal right jugular vein and superior vena cava. It was suggestive of malignant etiology like SVC leiomyosarcoma. A differential diagnosis of intimal sarcoma was also considered. The second case was a 53 years old female who came with the complaints of pain abdomen and weight loss. The CT scan revealed a lesion in Inferior vena cava, 2 cm proximal to IVC bifurcation and 6 cm distal to right renal vein. [Figure 1and 2] Both the patients underwent excision of the tumor with the vessel and reconstruction with a Dacron graft was done. The specimens were sent for histopathological examination. GROSS EXAMINATION The first case showed a segment of large vessel measuring 9.5x4.5x4 cm. A mass was seen attached to the inner wall of vessel and protruding outside through the lumen. Tumor on cut section was solid, firm, homogenous grayish white with focal necrotic areas. In the second case, the vessel wall with an attached firm to hard lesion was received. It measured 4x3.8x3.2 cm. It also showed some bony hard areas. [Figure 3 and 4] MICROSCOPIC EXAMINATION Microscopic features studied from multiple sections showed a cellular spindle cell tumor adherent to and present within the lumen of SVC. Tumor was composed of intersecting fascicles of spindle cells having pleomorphic, vesicular nuclei and moderate amount of eosinophilic cytoplasm. There were 25 mitosis per 10 high power field. Focal tumor necrosis was seen. Surgical margin was unremarkable. The second case also showed similar histological appearance with large areas of metaplastic bone with adjoining areas showing osteoclastic giant cells. [Figures 5-8] IMMUNOHISTOCHEMISTRY Tumor cells were diffusely positive for Smooth muscle actin and Desmin and negative for S100 and Cytokeratin. [Figures 9 and 10] DISCUSSION Primary vascular leiomyosarcomas are very rare malignant tumors.4 The tumor originates from proliferation of smooth muscle cells of the media and may grow intravascularly or extravascularly or both.5 Growth pattern of the tumor varies from intraluminal to extraluminal or both. About 62% of cases have extraluminal, 33% have both, and only 5% of cases have intraluminal growth pattern.6 Distant dissemination is rarely described. It involves large veins almost five times more than arteries, and the most common site is inferior vena cava and its branches.3 The SVC localization is extremely rare.7 IVC leiomyosarcoma is most frequently seen in the sixth decade with a female predominance.8     The diagnosis is often challenging due to rarity of the tumor and nonspecific complaints. The optimal treatment for IVC leiomyosarcoma is still not known as few cases have been reported. Complete wide resection of the tumor with adjunctive radiotherapy or chemotherapy is the most preferred treatment.9 Nonmyogenic sarcomas, which are derived from the intima, are even more infrequent and are typically seen in the arterial system, particularly the pulmonary artery.10 Intimal sarcomas have also been reported in the Superior vena cava, Inferior vena cava and brachiocephalic vein.11,12 Typically leiomyosarcoma and angiosarcoma carry a better prognosis than undifferentiated intimal sarcoma with a mean 5-year survival of 33–53%.13 Immunohistochemical examination reveals positivity for Smooth muscle actin and Desmin in one half to nearly 100% of tumors.14 In the present cases, tumor cells displayed nuclear atypia and IHC stains were positive for Smooth muscle actin and Desmin. CONCLUSION Leiomyosarcoma of the Superior and Inferior Vena Cava are rare tumors and often malignant. An accurate imaging and histopathological diagnosis is essential as they can affect the prognosis and treatment approach. Immunohistochemistry helps in differentiating it from other entities. CT scan helped in localizing the position of the tumor and also its relationship to the surrounding structures. Long term survival depends on obtaining an early diagnosis and performing an extensive surgical resection with tumor free margins. So such cases should be thoroughly examined and followed. 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 for this article has been reviewed and discussed. Conflict of interest: None Englishhttp://ijcrr.com/abstract.php?article_id=2510http://ijcrr.com/article_html.php?did=2510 Spaggiari, L., Regnard, J.F., Nottin, R. et al. Leiomyosarcoma of the superior vena cava. Ann Thorac Surg. 1996; 62: 274–276. Picquet, J., Blin, V., Dussaussoy, C. et al. Surgical reconstruction of the superior vena cava system: indications and results. Surgery. 2009; 145: 93–99 Chaumont A, Pierret C, Kerangal X, Le Moulec S, Laborde F. Leiomyosarcoma of the Superior Vena Cava. The Annals of Thoracic Surgery. 2014;98(2):725-727. Levett JM, Meffert WG, Strong WW, et al. Leiiomyosarcoma of the superior vena cava and azygous vein. Ann Thorac Surg 1995;60:1415-7. Tovar-Martin E, Tovar-Pardo AE, Marini M, et al. Intraluminal leiomyosarcoma of superior vena cava: a cause of superior vena cava syndrome. J Cardiovasc Surg 1997;38:33-5. Huang J, Liu Q, Lu JP, Wang F, Wang L, Jin AG. Primary intraluminal leiomyosarcoma of the inferior vena cava: Value of MRI with contrast-enhanced MR venography in diagnosis and treatment. Abdom Imaging 2011;36:337-41. Benvenuti M, Lorusso R, Gelsomino S, et al. Resection of a primary leiomyosarcoma of the superior vena cava and right atium on a beating heart. Int J Cardiol 2011;15:151-3. Hemant D, Krantikumar R, Amita J, Chawla A, Ranjeet N. Primary leiomyosarcoma of inferior vena cava, a rare entity: Imaging features. Australas Radiol 2001;45:448-51. Kieffer E, Alaoui M, Piette JC, Cacoub P, Chiche L. Leiomyosarcoma of the inferior vena cava: Experience in 22 cases. Ann Surg 2006;244:289-95.  Fujita H., Kawata K., Sawada T., et al. Rhabdomyosarcoma in the inferior vena cava with secondary Budd-Chiari syndrome. Internal Medicine. 1993;32(1):67–71.  Sebenik M, Ricci A,  Di Pasquale B, et al. Undifferentiated intimal sarcoma of large systemic blood vessels: report of 14 cases with immunohistochemical profile and review of the literature. The American Journal of Surgical Pathology. 2005;29(9):1184–1193. Rytina E. R. C., Govil Y. K., Sabanathan K., Ball R. Y. Intimal sarcoma of the right brachiocephalic vein presenting as the superior vena caval syndrome. Journal of Clinical Pathology. 1996;49(4):347–349.  Burke A. P., Virmani R. Sarcomas of the great vessels. A clinicopathologic study. Cancer. 1993;71(5):1761–1773. Weiss SW, Goldblum JR. Enzinger and Weiss’s soft tumor: 5th ed (Mosby Elsevier, Philadelphia, USA) 2008; 545-62.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411015EnglishN2018August16Life SciencesRecovery of Fingerprint Ridges from Crime Scene; Percentile of Actual Implemented Ridges Details in Each Quadrant Andits Feasibility   English1014Rhythm GandhiEnglish Amit ChauhanEnglish S. K. ShuklaEnglishAim: To determine the actual percentage of partial or complete fingerprints from each quadrant that are recovered from the crime scene. Methodology: In this study, 100 samples including male and female were collected from the population of Haryana from the age group 18-35 years at the temperature 18-350 C. All lateral fingerprints were collected on white A4 sheet by applying black fingerprints ink over the fingers. For examination of sample, 10 X lens and for statistical evaluation SPSS software latest version 17.0 was used. Result: It was observed that the females have similar number of ridges in thumb as the males had. Composure of the fingers have a slight bit variation present in the male study. Discussion: As the resultant of the present study, it was observed that thumb impression, little finger impression for both of genders shows the significant difference of their presence over any surface. The ridge details show that females have highly significant lesser number of ridges in comparison of males. In both of fingers (thumb and little finger) both of genders, females have high chances in which no. of ridges may be absence. Conclusion: This study may be useful to get an idea about the gender and to get an idea about the finger from which it may be implemented over the object.  EnglishFingerprints ridges, Gender discrimination, Crime scene, Percentage, Feasibility etcINTRODUCTION In last few decades, investigation and identification procedure to an individual has changed; the modern technology has taken over the traditional process of investigation(1) (3). In this era of advancement, since the digitalization has minimized time duration of process yet(2), we do the final submission after manual examination in few of forensic aspects i.e. fingerprints, questioned documents etc. fingerprints and palm prints which are perpetual, unique and ubiquitous by nature, i.e. play a vital role in identification of an individual and investigation(4) (5). These finger prints, palm prints carry tremendous information about suspect, gender, an approximation of the age. numerous Scientists and scholars carried out a lot of research over finger prints and palm prints over distinct populations(6). As per the existing studies of scientists, these prints are recovered in various forms and only few details are noticed. When someone writes, prepare of work of art, or take the support of wall and the ridge and furrows of the palm that have sweat pores which keep them moist. Individual put their hands against the surfaces, nobody help them to support or facilitate the movement of hands and unknowingly their identity left behind over the surface in latent form(7) (8). By this time, it has been observed that only a few ridges are encountered belong to any part of palmar surface(9). When an individual put their hand against any surface, the outer edge of thumb, tip point of index finger, while the middle portion of middle finger and ring finger comes in the contact of that surface. In case of little finger, it repeats the same manner like of thumb and only outer edge details are recovered from surfaces(10). It is frequently observed that partial information is obtained from the intensified prints and the identity left often in question. According to a research of federal Bureau of Investigation, the probability of any similarity in two fingerprints is about 1.9x1015which is very rare and have not reported yet. This study was conducted to obtain an information about the implementation of ridge details over any object and to determine the portion of any finger(11). Will the implementation give any discrimination between two genders? Or both of genders will have equal probability for implementation of their fingerprints ridges over any surface. For which all the ten fingerprints were divided in to four quadrants (1st, 2nd, 3rd and 4th quadrant) from the center of pattern because of any occurrence of ridge, only middle portion is recovered. METHODOLOGY In this study, 100 samples including male and female were collected from the population of Jharoda, Najafgarh areas of Haryana from the age group 18-35 years. All the samples were collected from March -April 2017 at the temperature in between 18-350 C. All the subjects were informed about the purpose of study and consent was taken earlier. Samples were collected by simple random sampling method(12). All the samples were preserved in simple A4 size Brown envelope at room temperature to avoid any kind of degradation such as moisture, dirt. To determine the percentile of samples, each fingerprint samples were divided into four quadrants from the center of pattern. Shown below in figure-1. All lateral fingerprints were collected on white A4 sheet by applying black fingerprints ink over the fingers. Subjects were asked to implement their fingerprints as such as it is put over any documents without following any specific manner. All the samples were preserved for further analysis procedure. For examination of sample, 10 X lens was used and all samples were photographed with the help of Samsung 7s model -13 mega-pixel camera. For statistical evaluation SPSS software latest version 17.0 was used. A hypothesis was established for the conclusion of result whether, the occurrence of all quadrants will implement in equal proportion or only a few ridges from any quadrant will present. RESULT This study is mainly focused to study the occurrence of quadrant of fingers which is confronted at scene. When a left- handed person takes support of wall, write over a writing surface or prepare work of art, the outer edge of thumb (2nd and 4th quadrant) comes in the contact of surface(13) (14). While in Index finger, all four quadrants have equal chance of occurrence, in Middle finger, ring and little finger have equal proportion of occurrence. The proportion of a male left-handed person’s ridge details are given below in table No.-1 below When the male individual is right-handed, in that case the chances of a finger quadrant occurrence changes. During the study of ridge detail of a thumb prints, the quadrant (1st and 4th) have most probability of occurrence while in rest of finger it is almost similar. The ridge details for a right handed subject is given below in table no-2. During this study, it was observed that the females have similar number of ridges in thumb as the males had. Composure of the fingers have a slight bit variation present in the male study. The dossier for left-handed female is given below in table no.-3 The dossier for right-handed female has some similarity like of right-handed males. The details are given below in table no-4- DISCUSSION According to this study, the males of both of handed show 100% chance of higher probability of ridge details in 2nd and 4th quadrant of left hand’s thumb while less probability in 1st and 3rdof right hand’s thumb(15). Whereas, only 4% population showed the presence of ridge details of fingerprints in 1st quadrant of thumb and only 6% population showed the presence of ridges in 3rd quadrant of thumb. For index finger, male showed 100% presence of 1st and 3rd quadrants whereas 6% population showed the absence of ridges in 2nd and 4th quadrant. In analogous manner, middle finger’s ridges showed 100%presence in 1st and 3rd quadrants present in the whole selected samples whereas only 4% population showed the absence of the 2nd and 4th quadrants. The ring finger shows the similar percentage of ridges as in middle finger, it was 100% of all the quadrants were present in the ring finger while for little finger 2% for 1st quadrant, 14% for 2nd quadrant, 4% for the 3rd and 14% for the 4th quadrant showed the absence, in the left hand. In the dossier of right-handed males, the percentage was only 2% in 2nd and 4th quadrant of samples in thumb while for 1st and 3rd quadrant 100 % occurrence was observed. For index finger, the presence of ridges in 1st and 3rd quadrant was 82% while in 2nd 100% ridges were present and in 4thquadrant 4% ridges were absence. In case of middle finger, 100% ridge details were observed in 1st,2nd and 3rd quadrant while in 4th quadrant only 6% ridges were absence. In 1st and 3rd quadrant of ring finger showed the presence of 100 % ridge details while in 2nd and 4th only 4% were absence. For little finger of right handed males, in 1st quadrant 2% ridges were absence while in 2nd and 4th quadrant shows 4% of absence and 6% ridges were absence in 3rd quadrant of fingers. According to the dossier of this study, there is 100% chance of showing higher probability of ridge details in 2nd and 4th quadrant of the right hand’s thumb while less probability; 2% only in 1st and no occurrence of 3rdquadrant of the right hand’s thumb. For index finger, male showed 96% and 94% presence of ridges in 1st and 3rd quadrants whereas 12% and 8% population showed the absence of ridges in 2nd and 4th quadrant. In analogous manner, middle finger’s ridges showed 100% presence in 1st and 3rd quadrants present in the whole selected samples whereas only 6%, 4% population showed the absence of the 2nd and 4th quadrants. The ring finger distinguish percentage of ridges as in middle finger, it was 96% for 1st quadrant, 94% for 2nd and 3rd quadrant and 100% for 4th quadrant. For little finger 10% for 1st quadrant, 10% for 2nd quadrant, 6% for the 3rd and 10% for the 4th quadrant showed the absence, in the left hand. In the dossier of right-handed females, the percentage of ridges were;98% and 100%% in 2nd and 4th quadrant of the samples in thumb while for 1st and 3rd quadrant 100 % occurrence was observed. For index finger, the presence of ridges in 1st and 3rd quadrant was 90% and 98%% while in 2nd90% ridges were present and in 4th quadrant 90% ridges were present. In case of middle finger, 94%, 98% and 92% ridge details were observed in 1st,2nd and 3rd quadrant while in 4th quadrant 100% ridges were present. In 1st and 3rd quadrant of ring finger showed the presence of 98 and 96% % ridge details while in 2nd and 4th only 2% and 8% were absence. For little finger of right handed males, in 1st quadrant 14% ridges were absence while in 2nd and 4th quadrant shows 2, 12%% of absence and 8% ridges were absence in 3rd quadrant of fingers. CONCLUSION Fingerprints, palm and sole prints are used to establish the identity of an individual since so long in the history of investigation(16). Implementation of tremendous change in traditional methods and advancement has changed the investigation procedure of crime scene. A lot of studies have been carried out for the gender discrimination over various population in India, although these studies dealt with the information obtained from the tip of fingers while at scene of occurrence only a part of prints is encountered(17). Often, the outer part of thumb impression and inner part of index finger is encountered in various forms are observed. If the ridges are examined carefully and the investigator could determine the hand/ finger to which the ridges belongs, it will be helpful for investigation and no suspect will be able to escape from nabbing. Identification can be done based-on minutiae details among the ridges selected in an area followed by international standard. It will further help the analysts to direct search a particular gender will eventually save the time of investigating officer. ACKNOWLEDGEMENT The Authors acknowledge the immense help received from the Amity Institute of Forensic Sciences that provide all the facilities for this work. The authors are also grateful to editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. ETHICAL CLEARANCE: NA SOURCE OF FUUDNING; NA CONFLICT OF INTEREST: NA Englishhttp://ijcrr.com/abstract.php?article_id=2511http://ijcrr.com/article_html.php?did=2511 Caplan R.M. How fingerprint came into use for personal identification. J Am Acad. Dermatol. Vol. 23 issue 1 (1990), Pp; 109-14. Subrahmanyam B.V. Personal Identity. Modi’s Medical Jurisprudence and Toxicology 22nd Edition. Butterworths, New Delhi. 2001, Pp; 37-90. Rutty GN, Stringer K, Turk EE. Electronic fingerprinting of the dead. International Journal of Legal Medicine. 2007 Feb 13; Epub. ahead of print. Amit Chauhan, Jyoti Singh, Identification of an individual from the latent palm prints present on documents International journal of Research Science and Innovation, Vol. I Issue VII, pp-29-35, 2014. Amit Chauhan, Jyoti Singh, K.P.S. Kushwaha An Evaluation; Sexing from the Ridge density of latent palm prints of North Indian population, Research Journal of Recent Science, Vol-4, Pp-73-75, 2015 . Wang Y, Hu J, Phillips D. A Fingerprint Orientation Model Based on 2D Fourier Expansion (FOMFE) and its application to Singular-Point Detection and Fingerprint Indexing. IEEE Trans Pattern Anal Mach Intell. Vol 29 Issue 4 (2007), Pp; 573-85. Jain A.K, Chen Y, Demirkus M. Pores and ridges: high-resolution fingerprint matching using level 3 features. IEEE Trans Pattern Anal Mach Intell. Vol. 29 issue 1 (2007) Pp; 15-27. Amit Chauhan, Aditi Chauhan, Dr. Jyoti Singh, Dr. S. K. Shukla. A correlative study between the implementation of rhythmic system and hieroglyphicssubstantial’s. International Journal of current research and review. Vol 9 Issue 9 (2017). Pg; 1-05. Amit Chauhan, Aparna Gautam, Sourabh Kumar Singh, Dr. S. K. Shukla. Gender inequity from the quadrant of lateral fingerprints among the age group of 18-25 years from the population of National capital region of India. International journal of civil engineering and technology. Vol. 8 Issue 4 (2017). Pg; 1402-1407. Lin SS, Yemelyanov KM, Pugh ENJR, Engheta N. Polarization-based and specular-reflection-based noncontact latent fingerprint imaging and lifting. J Opt Soc Am A Opt Image Sci Vis. Vol 23 issue 9 (2006), Pp; 2137-53. Acree Mark A. Is there a gender difference in fingerprint ridge density? Forensic Science International. Vol 102 (1999), Pp:35-44. Grieve MC, Dunlop J. A practical aspect of the Bayesian interpretation of fibre evidence. J Forensic Science Society. Vol. 32 (1992), Pp:169-75. Plato C.C, Cereglino JJ, Steinberg FS. The Dermatoglyphics of American Caucasian. Am J Phy Antrhop. Vol 42 (1975), Pp:192-210. Cummins H, Midlo C. Fingerprints, Palms and Soles. An introduction to dermatoglyphics, Dover Publ, New York, 1961:272. Amit Chauhan, Varsha Chauhan. An expansion of indented signatures over the credential by the employment of domiciliary commodity. International journal of civil engineering and technology. Vol 8 Issue 4 (2017). Pg; 1960-1966. Moore R. T. Automatic fingerprint identification systems. In H.C. Lee, R.E. Gaensslen (Eds.) Advances in Fingerprint Technology, CRC Press, Boca Raton, FL, 1994:169. Okajima M. Frequency of fork in epidermal ridge minutiae in fingerprint. Am J Phys Anthrop. Jan-May Vol 32 (1970), Pp:41-8.  

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411015EnglishN2018August16Life SciencesDevelopment of Polymerase Chain Reaction Assay for Targeting Cytochrome C Maturation F (ccmF) Gene of Leptospira   English1519Potdar Gayatri A.English Dhotre Dheeraj P.English Pol Sae S.English Bharadwaj Renu S.EnglishLeptospirosis is a bacterial zoonotic disease caused by spirochetes of the genus Leptospira. The direct method of diagnosis of Leptospirosis has been so far by culture isolation but isolation rate of the microorganism from clinical specimens is low. The additional conventional method is the detection of antibodies which is done by ELISA, that fails to identify the infecting serovar. MAT is used as the gold standard, but it can only confirm the disease at a later acute phase because anti-Leptospira antibodies usually become measurable only 5 to 7 days after onset of illness. Thus, serology does not contribute to early diagnosis of Leptospirosis. To overcome these limitations, we developed PCR assay targeting ccmF gene of Leptospires using in-house designed RGf and RGr primers, with a product size of 910bp. The protocols were standardized. PCR could detect the target bacterial gene without any ambiguity. This simple, cost-effective and rapid method can be applied to detect causative Leptospira interrogans serovars from the isolates.  English PCR, ccmF gene, Molecular detectionIntroduction: Leptospirosis is a disease that is caused by spirochetes, members of the genus Leptospira. It is well illustrated as the frequent zoonosis in the world. Leptospirosis is most common in the tropics and distributed worldwide (except in the Polar Regions) (1). Under reporting is a major concern in analyzing the actual incidence of leptospirosis in many Asian countries (2). In India, it is directly related to either monsoons or poor sanitary conditions, with multiple epidemics reported (3–6). For quick epidemiological response and also for timely treatment paradigms, rapid and simple assays that can reliably detect and distinguish Leptospira are necessary. Combining clinical proficiency and alertness with confirmatory laboratory back up noticeably increases the recognition of patients with Leptospirosis. As the clinical features are imprecise, laboratory test play a key role in diagnosis of Leptospirosis (7). Since isolation rate of the microorganism from clinical specimens is low, often due to the prior indiscriminate use of antibiotics, serological techniques remain the cornerstone of diagnosis. The analysis of acute- and convalescent-phase sera by ELISA and the microscopic agglutination test (MAT). MAT is used as the gold standard, but it and can only confirm the disease at a later acute phase because anti-Leptospira antibodies usually become measurable only 5 to 7 days after onset of illness. Hence, serology does not contribute to early diagnosis of Leptospirosis. Early diagnosis is obligatory because antibiotic treatment is most valuable when it is initiated early in the course of the disease. It is imperative to note that, in distinction to many of the resembling diseases (e.g. dengue), leptospirosis can easily be treated with antibiotics. This is provided that the diagnosis is confirmed in the first week of disease onset, when treatment with antibiotics is most effectual. Thus to facilitate preliminary treatment at the most effective time point, the availability of an accurate diagnostic test that is needed in the early acute phase of the disease. Alternative methods were developed to assess the presence of leptospires in clinical samples like immune peroxidase staining (8), Immuno fluorescence staining (9) and DNA hybridization (10). These were useful methods for the detection of Leptospira, provided that the micro-organisms should present in fairly large numbers. These were not satisfactory for routine diagnostic purposes, mainly because of their lack of sensitivity. Rapid recognition of small numbers of leptospires may become realistic due to precise amplification of Leptospiral DNA (11,12). PCR is a sensitive, specific, and rapid technique which has been successfully applied to the detection of several microorganisms and viruses in a variety of clinical specimens, including sputum, serum, cerebrospinal fluid, urine, faeces, and various tissues. PCR can rapidly confirm the detection in the early phase of the disease and before antibody titres are at detectable levels.           Several PCR assays have been developed for the detection of leptospires species, and that are unable to detect Leptospira at serovar level. It is epidemiologically important to identify the infecting Leptospira serovars, as they may be associated with certain animal host species. So the appropriate control measures can be instituted.           The aim of this study was to identify the gene with potential to enhance Leptospira serovar discrimination by sequencing-based methods. We used bioinformatics tools to check the discriminatory power of existing genes. Previously deposited sequence data were evaluated by phylogenetic analyses Materials and Methods: Computational determination of discriminatory powers of housekeeping genes            Thirteen housekeeping genes were selected in a present study by comparison between two Leptospira genomic sequences available in gene bank (http://www.ncbi.nlm.nih.gov) those of L. interrogans serovars Copenhageni strain Fiocruz L1-130 chromosome I (accession number AE016823.1) and L. interrogans serovar Lai strain 56601 chromosome I (accession number AE010300.1)            Multiple sequence alignments (MSA) for these sequences were carried out using CLUSTALX Version 2.1 software (13). Multiple Sequence Alignments were  manually edited to remove ambiguities using DAMBE software (14). Polymorphic sites were counted and percent polymorphism for each gene was determined (Table: 1). Primer Design: As ccmF gene was found to have maximum percent polymorphism (i. e. 5.8%), and was selected for further study. Primer 3 software  was used to design primers for the hyper variable region of ccmF gene (15). In order to ensure optimal primer pair, primer length was set 20 bp, Tm was set at 450C to 600C, and the difference between forward and reverse primer sets was minimized (within 20C). The target value for primer GC content was 50–60%. No secondary structures within primers were allowed. All above parameters were manually checked by Generunner (http://ftparmy.com/130936-gene-runner.html) software.          To check the lack of sequence homology with other bacterial or eukaryotic DNA sequences, designed primer sequences were checked for with a primer BLAST (NCBI, Bethesda, MD)(http://blast.ncbi.nlm.nih.gov/Blast.cgi) search. In silico designed primer pairs were synthesized by commercial manufacturer (Sci Fi biological) and were named as RGf and RGr primers. Utility of primers:  DNA extraction from standard serovars: Five Leptospira interrogans serovars were obtained from National Leptospirosis Reference Centre, Regional Medical Research Centre in Port Blair, Andaman and Nicobar islands (listed in table: 2). The serovars were grown in Ellinghausen–McCullough–Johnson–Harris (EMJH) media (BD, Sparks, MD) at 300C for 6–8 days. Leptospiral DNA was isolated from standard cultures with a Qiagen Blood and Cell Culture DNA Mini kit (Qiagen, Valencia, CA). PCR standardization:           A total reaction volume of 25 µL consisted of 10 µL Qiagen Master Mix, 1.5 µL of forward primer (2 pmol /µL stock), 1.5 µL of reverse primer (2 pmol /µL stock), 10 µL nuclease-free water and 2 µL DNA template. For negative controls, nuclease-free water was used instead of the DNA template. Samples were run for 2 min at 940C, followed by 30 cycles of 30 s at 94 0C, 30 s at 550C and 30 s at 72 0C on a Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA) At the end of the 30 cycles was an extension period of 7 min at 720C. PCR products were visualized on a 2% agarose gel and documented with a Gene Flash gel documentation system (Syngene, Frederick, MD). Each sample was run at least two times for reproducibility (Fig 1).           Gene amplicons were sequenced using ABI 3730xl and sequences were submitted to NCBI to get accession numbers (Table 3).  Phylogenetic tree of L. interrogans serovars for ccmF gene Multiple sequence alignment (MSA) for sequences of standard leptospira serovars was carried out using CLUSTALX Version 2.1 software. Multiple Sequence Alignment was manually edited to remove ambiguities using DAMBE software. Polymorphic sites were counted and percent polymorphism for ccmF gene was determined. Phylogenetic tree was constructed by Neighbour Joining  method using MEGA5 software package (16).  Specificity testing of  primers To check cross-annealing of primers with other species, ccmF primers designed were tested against the isolates of Bacillus cereus, Escherichia coli, Candida tropicalis, Salmonella typhi, Staphylococcus aureus and Mycobacterium tuberculosis. DNA was isolated from these cultures with a Qiagen Blood and Cell Culture DNA Mini kit, and were tested by ccmF gene PCR. Results: ccmF gene (encoding Cytochrome C biogenesis protein) had maximum percent polymorphism i.e 5.8% among other genes studied.  Thus it was selected for further studies and primers were designed and PCR was standardized. BLAST output of primers showed high degree of specificity for Leptospira species. The size of the amplification products corresponded to the expected fragment size i.e. 910 bp. The genetic distance between the L. interrogans serovars in the phylogenetic tree is represented by a bar which represents 0.005 nucleotide substitutions per alignment position among the serovars. This demonstrates variations in the sequence homology of ccmF gene which reflects in genetic diversity. All the Leptospira interrogans serovars under study were segregated from each other (Fig: 2).  Primer sets produced only amplification products for standard Leptospiral serovars and not the other test organisms. Discussion In the last two decades, several PCR assays for the detection of Leptospires have been illustrated. PCR should generate an amplicon with sequence variability appropriate for phylogenic assessment for molecular epidemiological purposes. The techniques such as, Restriction fragment length polymorphism analysis (17,18), ribotyping (17), pulsed-field gel electrophoresis (19) DNA hybridization (20), variable-number tandem-repeat analysis, arbitrarily primed PCR (APPCR), low-stringency single specific primer PCR (LSSP- PCR), PCR restriction endo- nuclease analysis (PCR-REA) (21) etc. are available for Leptospira serovar identification. However, fragment length polymorphism, ribotyping and pulsed-field gel electrophoresis are laborious, and time-intensive and require significant amounts of genomic DNA, whereas methods such as AP-PCR and LSSP-PCR result in complex banding patterns, DNA-DNA hybridisation is laborious and requires the use of radio-labelled isotopes. Hence cannot be used for routine diagnostic purposes. Here we described the use of bioinformatics to identify novel gene i.e ccmF gene which is having potential of discrimination between Leptospira serovars. PCR tests developed by other researchers also provide fast testing platforms, but the primers used can only differentiate between Leptospira species and have some disadvantages. Primers presented in this study provide the ability to detect and differentiate between Leptospira interrogans serovars, that will be especially useful in epidemiological studies as the source for disease outbreaks. ccmF gene codes for cytochrome c-type biogenesis protein CcmF. Cytochrome C is found in respiratory or photosynthetic electron transport chain, contain covalently bound heme. Bacterial C type cytochromes are extra cytoplasmic proteins and can be soluble and membrane bound. Presently we know 12 bacterial genes which control cytochrome C maturation ccm (ABCDEFGHI) res ABC and four genes that control redox state of the cell. ccmF gene is housekeeping gene,  present in pathogenic and non pathogenic Leptospira which codes for cytochrome c-type biogenesis protein ccmF. It has an average length of 650 amino acids. As this gene has a housekeeping function, hence it is less susceptible to some forms of lateral gene transfer (22) . The species L. interrogans is one of the dominant pathogen majorly found in the most infections related to Leptospirosis when compared to other pathogenic species like Leptospira borgpetersenii, Leptospira santarosai, Leptospira noguchii, Leptospira weilii, Leptospira kirschneri and Leptospira alexanderi. Cerqueira et al, 2009 demonstrated that the L. interrogans species is associated with severe human Leptospirosis, while the other strains like L. santarosai have shown their association with pigs and cattle. Hence, we first analyzed the various serovars related to species L. interrogans (23). The potential of this assay must be balanced by a limitation: As ccmF gene is housekeeping gene, so it is present in non pathogenic Leptospira also. Hence DNA sequencing will be needed to discriminate between pathogenic and non pathogenic Leptospira. Conclusion The ccmF encoding gene had higher nucleotide divergence that allowed identification and also the potential for the discrimination between closely related Leptospiral serovars. The ccmF gene PCR may also likely to detect other L. interrogans  serovars hence it may be valuable tool for early diagnosis of  Leptospires directly from biological samples with clinical suspicion of leptospirosis. This new PCR could be useful in epidemiological studies to identify the source for disease outbreaks. Acknowledgements  The authors gratefully acknowledge Dr.Yogesh Shouche of Microbial Culture Collection, National Centre for Cell Sciences, Pune, MS, India for performing sequencing of Leptospira serovars. We thank the Director, Dr.P. Vijayachari of National Leptospirosis Reference Centre, Regional Medical Research Centre (ICMR), Port Blair, India for providing us the leptospiral reference serovars. 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 this article has been reviewed and discussed. Conflict of interests The authors declare that they have no conflict of interests. Englishhttp://ijcrr.com/abstract.php?article_id=2512http://ijcrr.com/article_html.php?did=25121. Effler PV, Bogard AK, Domen HY, et al. Evaluation of eight rapid screening tests for acute leptospirosis in Hawaii. J Clin Microbiol.2002; 40: 1464-9. 2. Pappas G, Papadimitriou P, Siozopoulou V, et al. The globalization of leptospirosis: Worldwide incidence trends. Int J Infect Dis. 2008;12:351-7. 3. Sharma S, Vijayachari P, Sugunan AP, et al. Seroprevalence of leptospirosis among high-risk population of Andaman Islands, India. Am J Trop Med Hyg. 2006;74:278-83. 4. Manocha H, Ghoshal U, Singh SK, et al. Frequency of leptospirosis in patients of acute febrile illness in Uttar Pradesh. J Assoc Physicians India. 2004;52:623-5. 5.Karande S, Bhatt M, Kelkar A, et al. An observational study to detect leptospirosis in Mumbai, India, 2000. Arch Dis Child. 2003;88:1070-5. 6. Jena AB, Mohanty KC, Devadasan N. An outbreak of leptospirosis in Orissa, India: the importance of surveillance. Trop Med Int Health. 2004;9:1016-21. 7.Turner LH. Leptospirosis III. Maintenance, isolation and demonstration of leptospires.Trans R Soc Trop Med Hyg. 1970; 64: 623-646. 8.Terpstra WJ, Jabboury-Postema J, Korver H. Immunoperoxidase staining of leptospires in blood and urine. Zentralbl Bakteriol Mikrobiol Hyg. 1983; 254:534-539. 9.Terpstra WJ, Schoone GJ,  Schegget J. Detection of leptospiral DNA by nucleic acid hybridization with 32P- and biotin-labelled probes .J Med Microbiol. 1986; 22:23-28. 10. Van Eys GJJM, Gravekamp C, Gerritsen MJ, et al. Detection of leptospires in urine by polymerase chain reaction. J Clin Microbiol. 1989;27:2258-2262. 11.Mulli KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987; 155: 355-350. 12. Mullis K, Faloona F, Seharf S, et al. Specific amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986; 51:263-273. 13. Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0. Bioinformatics.2007; 23:2947-2948. 14. X. Xia and Z. Xie DAMBE: Software Package for Data Analysis in Molecular Biology and Evolution. J Hered. 2001; 92 (4): 371-373. 15. Untergasser A, Cutcutache I, Koressaar T, et al. Primer3 - new capabilities and interfaces. Nucleic Acids Research.2012; 40(15):e115. 16. Koichiro Tamura, Daniel Peterson, Nicholas Peterson, et al.MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods Mol Biol Evol. 2011 Oct; 28(1). 17. Perolat P, Merien F, Ellis WA, et al. Characterization of Leptospira isolates from serovar hardjo by ribotyping, arbitrarily primed PCR, and mapped restriction site polymorphisms. J Clin Microbiol.1994; 32: 1949–1957. 18.Kawabata H, Dancel LA, Villanueva SYAM, et al. flaB-polymerase chain reaction (flaB-PCR) and its restriction fragment length polymorphism (RFLP) analysis are an efficient tool for detection and identification of Leptos. 19.Hermann JL, Belleger E, Perolat P, et al . Pulsed-field gel electrophoresis of NotI digests of leptospiral DNA: a new rapid method of serovar identification. J Clin Microbiol.1992; 30: 1696–1702. 20. Zuerner RL and Bolin CA. Differentiation of Leptospira interrogans isolates by IS1500 hybridization and PCR assays. J Clin Microbiol.1997; 35: 2612–1617. 21.Brown PD and Levett PN. Differentiation of Leptospira species and serovars by PCR-restriction endonuclease analysis, arbitrarily primed PCR and low-stringency PCR. J Med Microbiol.1997; 46: 173–181. 22.Carsten Sanders, Serdar Turkarslan, Dong-Woo Lee, et al. The Cytochrome c Maturation Components CcmF, CcmH, and CcmI Form a Membrane-integral Multisubunit Heme Ligation Complex J Biol Chem. 2008 Oct 31; 283(44): 29. 23.Gustavo M. Cerqueira, Alan J. A. McBride, Mathieu Picardeau, et al. Distribution of the leptospiral immunoglobulin-like (lig) genes in pathogenic Leptospira species and application of ligB to typing leptospiral isolates. J Med Microbiol. 2009 Sep;58(Pt 9):1173-81.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-52411015EnglishN2018August16HealthcareFine Needle Aspiration Cytology [FNAC] – Review Article   English2025Sachin B. IngleEnglish Chitra R. Hinge (Ingle)EnglishFNAC (Fine Needle Aspiration Cytology) as we know it today dates back to around 1950. FNAC being easy, safe, cost effective should be preferred as first line diagnostic method by all clinicians. Before any surgical intervention, FNAC reports direct surgeon about what treatment modality to be used. Surgical pathology has its own confirmatory role post- operatively but importance of FNAC is also well known by all clinicians. Therefore, there has to be a setting of dedicated FNAC clinic in the department of pathology. With the help of imaging modalities, FNAC has evolved as more accurate and specific method, while the use of ancillary techniques makes even this easy procedure as highly useful for diagnosis and prognosis of various lesions.  English FNAC, Preoperative, Diagnostic testThe Origins of FNAC /Historical Aspects In mid –nineteenth century, Kun [1] (1847) and Lebert[2] (1851) and Menetrier[3] (1886) used  needles to  obtain cells and tissue fragments to diagnose malignant tumours. Kun described this technique as “new instrument for diagnosis of tumours”. Leyden [4] (1883) employed the same method to isolate pneumonic microorganisms. Grieg and Gray who in 1904 diagnosed trypanosomiasis in cervical lymph node aspirates from patients with sleeping sickness in Uganda. Their findings were reported by a captain Bruce in a British Medical Journal memorandum in 1904. In the mid -1920s there were attempts in New York and Chicago to employ large needle aspiration for a variety of sites ranging through lymph nodes, prostate and breast. In the UK in 1927, Dudgeon and Patrick [5] suggested the needling of tumours as a means of rapid microscopic diagnosis. Interest in the procedure was resurrected by Europeans in mid 1950s. It was in Europe that “FNAC” as the technique was usually called began to flourish in 1950s and1960s. Soderstrom [6] and Franzen [7] in Sweden, Lopes Cardazo [8, 9] in Holland, Zajdela [8] in France and others became major proponents, studying thousands of cases each year. FNAC soon established its place as a diagnostic routine to be used by team of pathologists and clinicians. History of FNAC has been very well documented by Grunze and Spriggs,[10] and Naylor [11]. FNAC as an important clinical TOOL FNAC is a simple, inexpensive, easily performed outpatient procedure which can provide a rapid diagnosis. It has been widely used in Europe for decades, mainly in Scandinavian countries [12-15] . A technique which is safe, rapid, relatively pain free, cost effective and accurate is always a clinician’s first choice  and this is what FNAC is about . It is eminently suitable as first line investigation for almost all superficial palpable swellings as well as many deep seated lesions. FNAC was initially conceived as a means to confirm a clinical suspicion of local recurrence or metastasis of known cancer without subjecting the patient to further surgical intervention. FNAC is a time tested simple office procedure having a high degree of diagnostic accuracy and precision. The specificity and sensitivity of diagnostic precision lie in range of 60% and 80% respectively. The acceptance both by surgeons and pathologists itself speaks of the tale of comfort which it allows. The art of medicine is practiced within a community of caregivers who are perched on innumerable speciality branches and these braches intersect each other at various times. Clinical consultations help to acquaint cytopathologist about probable diagnoses possible for any lesion. Often a major surgical biopsy can be avoided by performing a needle aspiration instead. FNAC SURPRISES THE SURGEON MANY TIMES Surgeons are always impressed by the help of FNAC to make diagnoses which affect treatment of patient in a wide manner. Many tumours being diagnosed high grade on FNAC make the surgeon to go for chemotherapy before surgical intervention. Such a simple technique and so many wonders. Benefits of FNAC are innumerable.  Cost effective It has lower risk than surgical biopsy. It is readily repeatable and useful for multifocal lesions. Minimal physical and psychological discomfort for the patient. Rapid reporting and bedside diagnosis of neoplastic, hyperplastic and inflammatory  masses. Active participation of patient in treatment planning and provides opportunity for fuller preoperative counselling. Elimination of a two stage procedure Therapeutic procedure for evacuation of cystic lesions. Allows cases to be  prioritized  when there is a waiting time for surgery  Permits the diagnosis of some benign conditions for which  there is no need for surgery It is a rapid means of confirmation and recurrence of previously treated malignancy without surgery.  Technique of FNAC (Fig1) Success of FNAC depends to a high degree on perfecting the technique of sampling and preparation of samples. Palpation skills learnt through practice and experience, judiciously complemented by radiological image guidance when appropriate are essential to obtain representative samples. Choice of needles, the use or not of aspiration and the manipulation of needle within the target relative to type of tissue decide the adequacy of sample. Finally, correct smearing, fixation and staining of samples is critical to assure optimal preservation and presentation of cells. Specific staining defines and highlights specific features of aspirate smears. Comparisons between two commonly used methods are Air drying followed by a Romanowsky type stain such as MGG. Alcohol fixation followed by H and E staining. Usually, pathologists trained in gynaecological cytology prefer alcohol fixed –Pap stained smears while those trained in haematology choose air dried MGG stained smears. Utility of special stains in diagnosing lesions PAS /diastase or alcian blue for mucins -  mucins can help in diagnosing mucin producing tumours of many anatomic sites (breast, gastrointestinal site, pancreas, ovary ) and sometimes can be completely devoid of cells (e.g. pseudomyxomaperitonei or pure mucinous carcinoma), yielding a false negative diagnosis. Prussian blue for Iron in hemosiderin containing lesions. Masson Fontana for melanin in melanoma Grimelius for argyrophilic granules Congo red for amyloid PAS  for glycogen –extracellular glycogen production is appreciated in tumours like  Ewing’s sarcoma, rhabdomyosarcoma , and glycogen rich clear cell tumours). Oil red O for fat Fouchet’s reagent counterstained with Sirius red  for bile pigments Microorganisms identified by Gram, PAS, ZN or Gomori silver stain.  Pitfalls or drawbacks of morphological diagnosis As every other technique in this world, FNAC also has some complications and limitations. These have been enlisted below: Instances of serious complications have been reported in relation to different sites and organs, such as major haemorrhage, septicaemia, vasovagal reaction, seeding of tumour, bile peritonitis, acute pancreatitis, pneumothorax etc[16]. Pre-operative FNAC may cause local tissue changes which could render subsequent histological diagnosis difficult .Such changes include hematoma, infarction, capsular pseudoinvasion, reparative reactions have been reported[17] Results and accuracy are highly dependent on quality of samples and smears. Many pathological processes are heterogeneous and tiny sample obtained from FNAC may not be representative. Some lesions are recognised on basis of  specific micro architectural pattern, which may not be represented in cytological preparations Small FNA sample may not allow full armamentarium of ancillary techniques to be drawn upon. Aspiration cytology requires highly skilled and trained personnel in both aspiration and assessment .Stewart commented in 1933 that “until the pathologist has familiarized himself with the various pitfalls, errors are certain to occur” and “it must not be inferred that the diagnosis is always a simple and that no errors have been made”. Extrinsic factors which may lead to diagnostic pitfalls are lack of or misleading clinical information, non-representative samples, contamination of samples by tissue adjacent to target lesion, artefacts due to poor processing of samples and too much reliance on and technical failure of ancillary tests. Intrinsic factors which may lead to diagnostic pitfalls arise mainly due to deviations from general cytodiagnostic criteria which can occur in various benign and malignant tumours. Pitfalls are an inseparable part of the practice of FNAC, but they can be minimised, if requisite diagnostic rigours are applied and care taken to correlate cytology with clinical an radiological findings. Judicious use of ancillary techniques also helps in reducing incidence of pitfalls. Ancillary techniques No wonder that technology is the fastest spreading tumour in pathology, but this tumour is having all gains. Various new techniques have revolutionised FNAC since its history. The pathologist must always keep in mind to apply any of these appropriate ancillary diagnostic techniques to cytological preparations. Electron microscopy –It is particularly useful in unusual lung or mediastinal lesions. Valuable information is obtained in recognizing neuroendocrine tumours, in specific diagnosis of melanoma, mesothelioma, and some carcinomas where immunocytochemistry often cannot provide such positive diagnostic features[18.19] . Immunocytochemistry- It is the most important recent development in diagnostic cytology. Monoclonal antisera to various proteins and cell products are nowadays commercially available.    Alcohol fixed smears are usually preferred over air dried smears. The avidin –biotin complex method is the most commonly used with both monoclonal and polyclonal primary antibodies. Diaminobenzidine is used as marker dye. Appropriate controls are crucial to achieve diagnostic accuracy[20]..The results of immunocytochemistry should be interpreted with caution in relation of conventional cytomorphology and clinical data [21].                                                                                                                                      Image analysis – there are 3 ways of image analysis Morphometry-quantitative analysis of geometric features of structures such as tissues, cells, nuclei or nucleoli [22,23]. B. Object counting – quantitation of mitosis or measurement of proliferation fraction of a cell population using antibodies. It also makes it possible to quantitate apoptotic figure by TUNEL assay [24]. C. Cytometry –based on ability to detect a particular substance of interest by a specific dye and to measure the concentration of dye by assessing optical density [25]. Fluorochromes can also be used such as propidium iodide dyes [26-28].  Powerful computers also have automatic cell classification based on pattern recognition for diagnostic [29,30], prognostic [31,32] and predictive purpose [33]. Quantitation of nuclear immunostain of  Estrogen and progesterone receptors [34], proliferation markers [35-37] can be done. Flow cytometry Based on fundamental work showing that DNA content , measured by UV visible light in unstained cells , double during cell cycle [38], followed by improved detection of antigens using fluorescence methods [39] and development of apparatus capable of counting [40] and sizing blood cells [41]. Molecular cytopathology Application of molecular probes to cytologic samples of human malignancies has refined the diagnostic and prognostic armamentarium [42-45]. In situ hybridization – It is a newly developed and global approach to detect genetic changes in tumors. For localization of specific nucleic acid within individual cells based on complementary binding of a nucleotide probe, labelled with non-isotopic reporter molecule, to a special target sequence of DNA or RNA [46]. Using probes to chromosome specific sequence, it is possible to detect aneuploidy in interphase nuclei [47-48] and losses, gains or amplification of chromosome regions with known prognostic value [49-50]. In situ amplification – based on PCR this allows recovery of large amount of DNA from minute quantities of starting material [51]. Various adaptations of PCR have been developed for cytological preparations  [52] such as PCR in situ hybridisation ,in situ PCR, reverse transcriptase in situ PCR[53], methylation specific PCR [54],and primed in situ synthesis [55] . The most crucial steps in optimising in situ amplifications are fixation and preparation of cells.  IMAGING METHODS FOR GUIDANCE OF ASPIRATION CYTOLOGY Nowadays, to make FNAC more accurate and precise, imaging modalities have been used for guiding the tract of needle. Various imaging modalities used are Fluoroscopy – Uses- Quick alternative for radiologist not experienced in USG guidance. Is most useful in guidance for small, very mobile lesions. Efficient sampling options for cortical bony lesions. Ultrasound – Only real time guidance which allows imaging in any plane and is only suitable guidance for biopsy of foetal tissues. Some parts of body [56] such as chest wall, musculoskeletal system, through neglected in past, have undergone an increase in interest for both diagnosis and interventional studies. CT scanning – localization of needle tip with in a lesion is very accurate with CT. There are very few areas of body which cannot be biopsied under CT control and extremely small lesions can be sampled. MRI – its sensitivity is generally greater than that of other imaging methods, particularly useful in brain, liver and breast [57]. Conclusion –Fine needle aspiration cytology has an utmost importance in the current era of surgical practice in the preoperative stage as it guides the clinician a lot in the treatment plan and mostly clear the pathological aspects of the disease avoid untoward complications related to disease and treatment for the sake of pathological diagnosis. Many times it avoids unnecessary surgical intervention Acknowledgement- Dr Ingle is grateful to the past and present members of his laboratory for their contributions to his studies. He is very much thankful to his journal team of IJCRR for invaluable suggestions and guidelines throughout the publication process of the manuscript. 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