We report a case of malignant melanoma (MM) metastatic to the parotid gland, initially discovered on fine needle aspiration (FNA). The patient presented with a mass in the parotid gland area with previous history only significant for prostatic carcinoma. The initial FNA impression was melanoma. The smears were hypercellular with bloody necrotic background. The cells were epithelioid with mild nuclear atypia. Discrete cytoplasmic pigmentation was seen. No lymphoglandular bodies were noticed. Fragments of benign salivary gland were also identified. The cytological diagnosis of MM triggered onsite thorough physical examination for potential primary, where a scalp pigmented lesion was discovered hidden by overlying covering hair. Our differential diagnosis included melanoma, metastatic carcinoma, and lymphoma. Further work up for melanoma with S100, HMB45, and Mart 1 confirmed our top differential diagnosis. We emphasize thorough physical examination in such circumstances, and the importance of onsite evaluation guiding clinicians looking for primary.

Fine needle aspiration (FNA) in the operating room is a convenient optional diagnostic approach. Our objective of this study was to evaluate the accuracy and study the potential benefits of fine needle aspiration in the operating room. Retrospective review of all intraoperative FNA that were requested and performed by pathologists over 20-month period was carried out. Immediate smears were interpreted by the cytopathology team after staining with Diff Quik stain. All cases were reviewed and correlation with subsequent tissue diagnosis was done. Accuracy was calculated and potential benefits were discussed. The number of aspirates was 32. Adequate material for immediate and final interpretation was achieved in 31 cases (sensitivity 97%). In 20 cases (63%), malignancy was diagnosed while a benign diagnosis was rendered in 12 (37%). All cases had a follow up tissue diagnosis. No false-positive cases were identified (specificity 100%). The information gained from this approach was considered helpful to the surgeons. We concluded that FNA of head and neck masses in the operating room is an accurate and sensitive diagnostic approach. The service provides helpful information to surgeons and is an additional optional diagnostic approach.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第20295号 / 人健博第43号 / 新制||人健||4(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 高桑 徹也, 教授 藤井 康友, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DGAM

A dissertation submitted to the Faculty of Health Sciences, University of Witwatersrand, in fulfilment of the requirements for the degree of Master of Science (Medicine) in the branch of Anatomical Pathology Johannesburg, 2012 / Objective To determine the effectiveness of the cell block technique for immunocytochemical diagnosis by comparing cytomorphologic preservation and immunocytochemistry (ICC) stains in paired cell block and conventional fine needle aspiration (FNA) samples. Study Design This was a prospective study. Material for both conventional smears and cell blocks were collected simultaneously during fine needle aspiration of 50 lesions comprising lymph node, lung and liver masses. Grading of cellularity, morphological preservation, architectural preservation, immunocytochemical staining intensity and presence of background staining were compared on paired FNA smears and cell block samples derived from the same case. Each arm of the paired analysis was performed blindly without knowledge of the grading outcome of the other. The Kappa statistic (Κ) was used to measure inter-rater agreement. Results The fifty samples evaluated included FNAs from the lung, 24/50 (48%); liver, 23/50 (46%) and lymph node, 3/50 (6%). The immunocytochemistry stains consisted of 44/50 (88%) CK7, 44/50 (88%) CK20, 18/50 (36%) TTF1, 10/50 (20%) synaptophysin, 10/50 (20%) Hepar-1 and 7/50 (14%) AE1/3. There was no overall agreement in preservation of cytomorphological detail and ICC staining between the two methods. The Papanicolaou stained conventional FNA smears fared better then cell block for the vi evaluation of nuclear and cytomorphologic characteristics; cells in the cell block were poorly preserved in many cases. The ICC stains worked better on the cell block samples due to lack of background and aberrant staining. Conclusion Conventional FNA smears and cell blocks complement each other. Our results indicate that it would be optimal to use both modalities in the diagnostic work-up of mass lesions amenable to FNA diagnosis; the former to assess morphology, and the latter for optimal immunocytochemistry results. In resource constrained settings, the cost implications of performing both conventional and blocked smears on all FNA material warrants further evaluation.

Skeletal muscle metastasis from breast carcinoma is a relatively rare clinical entity. We report two cases of breast cancer metastatic to the skeletal muscle, diagnosed by ultrasound guided fine needle aspiration (US-FNA) biopsy done by interventional cytopathologists at an outpatient cytopathology center. Our two patients presented with lower anterior neck firmness and chest wall mass, respectively. Ultrasound evaluation of our first case demonstrated hypo-echoic thickened anterior strap muscles while in the second case there was significant distortion of the anatomy from previous surgeries. It was necessary to proceed with FNA biopsy even when their ultrasound findings were equivocal, to establish a definite rapid diagnosis. The immediate onsite evaluation findings were suggestive of malignancy in both cases with subsequent core biopsy confirming the diagnosis of metastatic breast carcinoma. In cytopathology, point-of-care (POC) ultrasound is used as an adjunct tool that offers visual guidance during FNA of nonpalpable masses and enables sampling of lesional “hot” spots to ensure specimen adequacy. Studies have demonstrated a reduction in FNA nondiagnostic rates with the use of ultrasound-guidance consequently reducing health care costs associated with nondiagnostic FNAs. US-FNA also provides adequate samples for cell block preparations. Metastatic lobular carcinoma of the breast has a wide range of clinical presentations and a high level of suspicion is advised. Cytopathologists-performed US-FNA is a proven, less-invasive, cost-effective tool that provides timely cytologic diagnosis.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19929号 / 医博第4149号 / 新制||医||1017(附属図書館) / 33015 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 坂井 義治, 教授 森田 智視 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

There is an urgent need to develop ammonia removal treatment systems for municipal and industrial wastewater treatment due to the increasingly stringent ammonia effluent discharge regulations implemented by Canada, the United States, and the European Union. The objective of this dissertation is to develop new understanding and advance the current design and operation of total ammonia nitrogen (TAN) removal via the moving bed biofilm reactor technology (MBBR) for municipal and industrial wastewaters. The first specific objective is to develop a passive, low operationally intensive, efficient and robust design strategy for municipal wastewater treatment to achieve partial nitritation (PN) as a pre-treatment to anammox treatment without using control strategies such as operating at low dissolved oxygen, or the use of inhibitors. This first objective includes developing new knowledge of the biofilm, biomass and microbiome of attached growth PN systems. The second specific objective is to investigate the impact of defining a maximum biofilm thickness, via bio-carrier design, to enhance the effects of free nitrous acid inhibition for PN of municipal wastewaters. The third objective is to investigate the effect of influent copper concentration on nitrifying MBBR systems over long-term operations, to demonstrate the feasibility of the nitrifying MBBR as a solution for TAN removal from gold mining wastewaters. The results pertaining to the first objective, achieved via a study investigating the operation of a nitrifying moving bed biofilm reactor at elevated TAN surface area loading rates (SALRs) of 3, 4, 5, and 6.5 g TAN/m²∙d with the aim of achieving passive PN, demonstrates that operating at a TAN SALR value of 6.5 g TAN/m²∙d can achieve PN without restricting dissolved oxygen or using inhibitors. Operating at a TAN SALR value of 6.5 g TAN/m²∙d achieves a TAN surface area removal rate (SARR) of 3.5 g TAN/m²∙d, and a nitrite accumulation of 99.8% of the oxidized TAN, demonstrating the suppression of nitrite oxidizing bacteria (NOB) activity, while achieving elevated TAN SARR values. At the molecular-scale, there is a statistically significant change in the ammonia oxidizing bacteria (AOB) to NOB ratio from 1:2.6 to 8.7:1 as the TAN SALR increases from 3 to 6.5 g TAN/m²∙d; however, even at a TAN SALR value of 6.5 g TAN/m²∙d there is an NOB abundance of approximately 2%; thus demonstrating that NOB remain present in the biofilm, while their activity is suppressed by operation at elevated TAN SALR values. Furthermore, this system was shown to achieve stable PN consistently for over a period of 10 months of operation, demonstrating a robust, passive, low operational strategy for attached growth PN. The second objective of this dissertation is addressed through a study that compared the carrier design of defined maximal biofilm thickness (z-prototype carrier) to undefined maximal biofilm thickness (chip-prototype carrier) for PN via free nitrous acid inhibition of tertiary, low carbon, municipal wastewaters. The study demonstrates that defined maximal biofilm thickness is a preferred design choice to achieve attached growth PN. The chip-prototype carrier shows biofilm thicknesses and biofilm mass values that are ten-fold higher than the z-prototype carrier, which is shown to contribute to the impact of free nitrous acid on AOB and NOB activities. The z-prototype carrier shows PN is achieved after 3 hours of exposure to free nitrous acid while the chip-prototype carrier does not achieve PN within this same time of exposure. Therefore, the defined maximal biofilm thickness carrier is identified in this research as the preferred design option to achieve attached growth PN for municipal, low carbon, tertiary wastewater treatment. The results of the third objective, achieved via a study investigating the effects of influent copper concentrations on nitrifying MBBR during long term operations to gold mining wastewaters, demonstrates that there is no AOB inhibition in attached growth systems exposed to 0.1, 0.3, 0.45, and 0.6 mg Cu/L for long exposure times. A trend of increasing nitrite accumulation with increasing influent copper concentrations is shown, indicating that NOB inhibition occurs at influent copper concentrations of 0.3 mg Cu/L and greater, with the greatest NOB inhibition observed with an influent copper concentration of 0.6 mg/L. There is no statistically significant difference in biofilm characteristics at the copper concentrations tested; however, there is a trend of increasing biofilm thickness and biofilm roughness with increasing copper concentrations. This study demonstrates the resilience of the nitrifying biofilm to copper inhibition and demonstrates that the nitrifying MBBR is a promising system for removing TAN in mining wastewater in the presence of copper.

Στις αρχές της δεκαετίας του 80 μια νέα εποχή ξεκινά για την Μαστογραφία και τον καρκίνο του Μαστού, με τη χρήση της τεχνικής "χαμηλής δόσης Μαστογραφίας", τη σωστή ενημέρωση των γυναικών για την πρόληψη του καρκίνου του μαστού και την εφαρμογή του πληθυσμιακού ελέγχου σε ασυμπτωματικές γυναίκες, με συνέπεια να αυξήσουν τον αριθμό των μη-ψηλαφητών και μη ψηλαφητών καρκίνων μαστού που διαγιγνώσκονται σε πρώιμα στάδια (0, Ι, ΙΙ, ΙΙΙΑ κλπ) με καλλίτερη πρόγνωση και θεραπεία. Προηγούμενες κλινικές μελέτες προσπάθησαν να συσχετίσουν τα ακτινολογικά με τα ιστολογικά χαρακτηριστικά του καρκίνου του μαστού αλλά δεν κατάφεραν να αναδείξουν σημαντικές διαφορές όσον αφορά τα ιστολογικά και βιολογικά χαρακτηριστικά μεταξύ των συμπωματικών και των μαστογραφικά εντοπιζόμενων μη ψηλαφητών καρκινωμάτων του μαστού . Επιπλέον, αρκετοί ερευνητές φαίνεται να καταλήγουν στο συμπέρασμα ότι τα καλά προγνωστικά χαρακτηριστικά των καρκινωμάτων του μαστού που αναδεικνύονται με την προληπτική μαστογραφία σχετίζονται άμεσα ή έμμεσα με την επίδραση βιολογικών παραγόντων. Σκοπός της παρούσας μελέτης είναι η ανάδειξη των δεικτών που σχετίζονται με την μαστογραφική απεικόνιση της κακοήθων ογκόμορφων αλλοιώσεων ψηλαφητών και μη και της επιθετικότητας της νόσου. Μελετήθηκαν συνολικά πεντακόσιες εβδομήντα τέσσερεις ογκόμορφες αλλοιώσεις ψηλαφητές ή μη του μαστού σε πεντακόσιες εβδομήντα ασθενείς που εντοπίστηκαν κατά τη διάρκεια του μαστογραφικού ελέγχου στο εργαστήριο μας κατά την περίοδο 1994- 2004. Όλοι οι ασθενείς υποβλήθηκαν σε βιοψία δια λεπτής βελόνης (FNA) ή κατευθυνόμενη από με μαστογραφία χειρουργική βιοψία. Η ιστολογική εξέταση ανέδειξε 410/574 (71,4%) κακοήθεις ογκόμορφες αλλοιώσεις και 164/574 (28,5%) καλοήθεις. Ανοσοϊστοχημεία πραγματοποιήθηκε σε τομές παραφίνης σε 390 από τις 410 κακοήθεις αλλοιώσεις χρησιμοποιώντας μία ποικιλία μονοκλωνικών και πολυκλωνικών αντισωμάτων ενάντια στις εξής πρωτεϊνες: ER, PR, p53, HER-2,Ki 67 και KATH D. Οι κακοήθεις ογκόμορφες αλλοιώσεις ταξινομήθηκα από την Μαστογραφική τους απεικόνιση (σύμφωνα Breast Imaging Reporting and Dada System BI RADS) σε τρείς κατηγορίες : στην κατηγορία Β (οι αστεροειδείς κακοήθεις ογκόμορφες αλλοιώσεις) ήταν το (44,8%), στην κατηγορία Α (οι άτονες με ασαφή ή εν μέρει ασαφή όρια) το 36% και στην κατηγορία Γ (σκιάσεις και αποτιτανώσεις) που ήταν 18,2% όλων των κακοήθων ογκόμορφων αλλοιώσεων. Η ανάλυση των αποτελεσμάτων μεταξύ της μαστογραφικής απεικόνισης των κακοήθων ογκόμορφων αλλοιώσεων και των ιστολογικών και βιολογικών χαρακτηριστικών των ανέδειξε : Σημαντική συσχέτιση της μαστογραφικής απεικόνισης με το μέγεθος των ογκόμορφων αλλοιώσεων (p=0.01<0.05). Σημαντική συσχέτιση με το βαθμό διαφοροποιήσεως της κακοήθειας (p=0.005<0.05). Σημαντική συσχέτιση με την έκφραση των πρωτεϊνών p53 (p=0.015) και Ki -67( p=0.02). Δεν παρατηρήθηκε σημαντική συσχέτιση της απεικόνισης των ογκόμορφων αλλοιώσεων με την ηλικία των ασθενών (p=0.08>0.05), με την ανίχνευση πυρηνικής θετικότητας για τους οιστρογονικούς (ER) και τους προγεστερονικούς(PR) υποδοχείς (p =0.4>0.05) καθώς και με τις πρωτεΐνες HER-2. και KATH D. Επίσης παρατηρήθηκε οριακή συσχέτιση στην απεικόνιση των κακοήθων ογκόμορφων αλλοιώσεων και των λεμφαδένων. Συμπερασματικά βρήκαμε ότι η κατηγορία Β με τις αστεροειδείς ογκόμορφες αλλοιώσεις είχε ευνοϊκότερη επίδραση από τ η κατηγορία Α με τις άτονες με ασαφή ή εν μέρει ασαφή όρια και με τις ογκομορφές αλλοιώσεις και αποτιτανώσεις (κατηγορία Γ). Οι άτονες με ασαφή ή με εν μέρει ασαφή όρια κακοήθεις ογκόμορφες αλλοιώσεις(κατηγορία Α) σχετίζονται με χαμηλής διαφοροποίηση κακοήθεια (grade 3) με υπερέκφραση του p53 και Ki -67 και με αρνητικούς υποδοχείς (PR και ER). Οι όγκοι αυτοί είναι μεγάλοι όγκοι με ίδια πολλές φορές μαστογραφική απεικόνιση με εκείνη των καλοηθών ογκόμορφων αλλοιώσεων. / At the beginning of 80 a new era for the Mammography and the breast cancer started with the use of the technique "low dose Mammography", with the correct information of women for the prevention of the breast cancer and the application of mass screening in asymptomatic women, with the result to increase the number of palpable and no- palpable breast cancers diagnosed in early stages (0,I,II,IIIA etr) with better prognosis and therapy. Previous clinical studies have attempted to correlate the radiological with the histological characteristics of breast cancer but did not succeed to show significant differences as for us the histological and biological characteristics among the symptomatic and the mammographically localized non- palpable breast cancers. Furthermore, many investigators see to conclude that the best prognostic characteristics of breast cancers that are identified with the preventive mammography are directly or indirectly related by the effect of biological factors. The aim of the present study is to mark out indicators that are related with the mammographic appearance of malignant palpable and no- palpable lesions and the aggressiveness of disease. In total five hundred seventy four palpable and no- palpable breast lesions have been studied in five hundred seventy patients that were localized during the mammographic screening in our department, between 1994 to 2004. All the patients were subjered to line needle biopsy (FNA) or mammographically guided open surgical biopsy. Histological examination showed 410/574 (71.4%) malignant lesions and 164/574 (28.5%) benign lesions. Immunohistochemistry was performed in paraffin sections in 390 of 410 malignant lesions using a variety of monoclonal and polyclonal antibodies against the following proteins ER,PR, P53, HER-2, Ki67and KATH D. The malignant lesions were classified from their mammographic appearance (according to Breast Imaging Reporting and Dada System BI RADS) in three categories: category A (lesions with poorly defined or partially poorly defined margins)were 36%, category B (speculated malignant lesions) were 44.8% and category C (lesions wih calcifications) were 18.2% of all malignant lesions. The analysis of our results between the mammographic appearance of the malignant lesions and their histological and biological characteristic showed the following: 1) Significant correlation existed between the mammographic appearance and the size of the lesions (p=0.01<0.05). 2) Significant correlation existed with the degree of differentiaton of malignancy (p=0.005<0.05). 3) Significant correlation existed with over expression of proteins P53 (p=0.015). and Ki- 67 (p=0.02). No significant correlation was observed between the appearance of lesins with the age of the patients (p=0.08>0.05), with the detection of nuclear positivity of the estrogens (ER) and progesterone (PR) receptors (p=0.4>0.05) and also with the proteins HER-2 and KATH D. Furthermore a bode line correlation was observed between the appearance of the malignant lesions and lymph notes. In summary, it was found that the category B patients with the speculated lesions had a more favorable effect than category A with the poorly defined or partially poorly defined limits, and the category C lesions with calcifications. The poorly defined or partially poorly defined limits malignant lesions (category A) are related with low grade malignancy (grade 3, with over expression of P53 and Ki-67 and with negative receptors (PR and ER). These are large size lesions and are difficulty diagnosed from benign lesions.

The work described in this dissertation focuses on developing simple yet effective assays integrating nucleic acid (NA) aptamers with rolling circle amplification (RCA) for the detection of non-NA biomarkers. The first project, a comprehensive literature review, highlights the current state of the art in functional NA-based RCA applications, and identifies shortcomings in the detection of non-NA targets with RCA. Biosensor design is critically evaluated from four key perspectives: regulation, efficiency, and detection of RCA, and the integration of all three components for point of care (POC) applications. The second project investigates how target-binding to a linear aptamer can be utilized to regulate RCA in a simple and inexpensive format. Phi29 DNA polymerase (DP) exhibits difficulty processing DNA strands that are bound to non-NA materials such as proteins. The work uses this restriction of phi29 DP as a feature by utilizing protein-binding aptamers as primer strands (aptaprimers) for RCA. The simplicity is showcased by adapting the method to a cellulose paper-based device for the real-time detection and quantification of PDGF or thrombin within minutes. As the second project is a turn-off sensor, the third project exploits the inherent 3’-exonuclease activity of phi29 DP to generate a simple turn-on assay instead. As target-bound aptamers were shown to be resistant to exonuclease activity, the phi29 DP preferentially digests target-free aptaprimers instead of target-bound aptaprimers. The target-bound aptaprimer could be liberated by a circular template (CT) by incorporating toehold-mediated strand displacement (TMSD), and used for RCA. Sensitivity was improved relative to project two, though the dynamic range was narrow owing to difficulty liberating target-bound aptaprimer at high target concentrations. Project four instead used RecJ, which has 5’-exonuclease activity, to modulate aptaprimer availability. Similarly to project three, target-binding conferred protection on the aptaprimer from 5’-exonuclease digestion by RecJ. By including a free 3’ terminus on the aptaprimer, inhibition of RCA due to target binding was avoided and CT-mediated TMSD was not needed, simplifying the assay. As well, this approach was generalizable as it was demonstrated using both a protein (thrombin) and a small molecule (ochratoxin A) target. This turn-on method further improved the assay compared to project three with a 100-fold enhancement in sensitivity and a restoration of the dynamic range. In sum, this work contributed multiple simple and sensitive approaches for the real-time fluorescent detection of proteins and small molecules with the RCA of linear aptamers. / Thesis / Doctor of Science (PhD)

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