Vid cytologisk bedömning av celler i exsudat kan olika metoder användas vid preparering. Vid konventionell utstryksmetod stryks provet ut manuellt på objektglas medan vätskebaserad ThinPrepmetod preparerar provet i en automatiserad process. Studiens syfte var att jämföra konventionell utstryksmetod och vätskebaserad ThinPrepmetod med tillsats av ättiksyra vid preparering av etanolfixerade exsudat. I studien användes totalt 34 prover av pleuravätska och ascites. Proverna preparerades med konventionell utstryksmetod och två varianter av ThinPrepmetod, ThinPrepmetod 1 och ThinPrepmetod 2. Preparaten bedömdes av två cytodiagnostiker utifrån fem kriterier; cellförekomst, förekomst av inflammatoriska celler, cellmorfologi, bakgrundsmaterial och förekomst av atypiska eller maligna celler. Resultaten visade på att ThinPrepmetod 2 gav likvärdiga, och i vissa avseenden bättre, resultat än konventionell utstryksmetod. ThinPrepmetoden har generellt fler fördelar jämfört med konventionell utstryksmetod, framförallt innebär den en tidsbesparing för de som bedömer preparaten vilket också gynnar patienter. En mer omfattande studie rekommenderas för att konfirmera resultaten i denna studie. / Preparation of effusions for cytological evaluation can be performed with different methods. Conventional smears are performed by spreading the sample directly onto a slide while liquid-based ThinPrep method prepares the sample in an automated process. The aim of this study was to compare conventional smears with liquid-based ThinPrep preparation, with additional acetic acid, of effusions fixated with ethanol. A total of 34 samples of pleural effusions and ascites were included. The samples were prepared with conventional smears and two versions of the ThinPrep method, ThinPrep method 1 and ThinPrep method 2. The preparations were examined by two cytotechnologists based on five criteria; occurrence of cells, occurrence of inflammatory cells, cell morphology, background material and the presence of atypical or malignant cells. The results showed that ThinPrep method 2 obtained equivalent and, in some aspects, even better results compared to conventional smears. In general, the ThinPrep method has several advantages compared to the conventional smears. In particular, it is timesaving when examining the slides, which also benefits the patients. A more comprehensive study is recommended to confirm the results of this study.

Urinblåsecancer är den sjunde vanligaste cancerformen i Sverige där blåssköljvätska utgör det huvudsakliga provmaterialet vid utredning. ThinPrep® är en vanligt förkommande metod som används inom vätskebaserad cytologi vid analys av blåssköljvätska. Fixering av provmaterialet kan försvåra diagnostiken på grund av förändrad morfologi samt artefakter.  Studiens syfte var att jämföra erhållet preparatinnehåll från blåssköljvätskor som fixerats med fixeringslösning innehållande ättiksyra med CytoLyt® solution. Studien ämnade även jämföra ThinPrep® filtren ”Non-GYN” och ”UroCyte”.  Studien inkluderade 90 patientprover av blåssköljvätska som fixerades med tillsats av ättiksyra samt CytoLyt®, varvid filtren ”Non-GYN” samt ”UroCyte” användes. Preparaten bedömdes av cytodiagnostiker samt cytopatolog utifrån kriterierna: bakgrundsmaterial, cellmängd samt färgbarhet. Resultatet visade att CytoLyt® i kombination med filtret ”Non-GYN” gav ett mer representativt preparat samt lämnade en god bakgrundsbild. Användandet av CytoLyt® resulterade i fler diagnostiska fördelar jämfört med fixering av ättiksyra, vilket även gynnar patienter. / Bladder cancer is the seventh most common form of cancer in Sweden where the main source of material for investigation are bladder washings. A common method within liquid-based cytology for analysis of bladder washings is ThinPrep®. Altered morphology as well as artifacts due to fixation can complicate funkar “the diagnostics of the sample. The aim of this study was to compare the slide content obtained from bladder washings fixed with fixative containing acetic acid with CytoLyt® solution. The study also intended to compare the ThinPrep® filters "Non-GYN" and "UroCyte". A total of 90 samples of bladder washings from patients where included. The samples where fixed with the addition of acetic acid and CytoLyt®, using the "Non-GYN" and "UroCyte" filters. A cytotechnologist and a cytopathologist assessed the preparations based on the criteria background material, cell quantity and the amount of colour change. The results showed that CytoLyt® in combination with the filter "Non-GYN" gave a more representative preparation and provided a good background image. The use of CytoLyt® resulted in more diagnostic benefits compared to fixation with acetic acid, which also benefits patients.

Immunhistokemi (IHC) och immuncytokemi (ICC) är viktiga verktyg inom cancerdiagnostik för att fastställa diagnoser. Både histologiskt och cytologiskt material kan användas för immunokemisk analys men kräver olika förberedelser. Den preanalytiska fasen påverkar epitop och antigenens tillgänglighet samt immunoreaktivitet, vilket gör valet av pre- och postfixering och hur de påverkar cytologiskt material, viktiga att förstå. ICC tillämpas vanligen på cytologiskt material som preparerats enligt cellblockteknik (CBT) men ibland saknas extraherat material för analys. Antikroppar kan användas för att differentiera tumörursprung, tumörsubtyper och inflammatoriska tillstånd, vilket är avgörande för klinisk diagnostik och behandlingsbeslut för individen. Vid klinisk patologi och cytologi i Kalmar är ett återkommande problem bristen på cellmaterial för CBT-preparering vid punktionscytologi. Syftet med denna studie var att validera antikroppar (kalretinin, CD45, CK5, CK7, SOX-10, EpCAM och TTF-1) på cytologiskt material (pleuraexsudat från 10 patienter) som preparerats enligt ThinPrep™- metoden (TP-metoden). Studien syftade även till att undersöka effekterna av post-fixering i 4 % formalin, 96% etanol eller ingen post-fixering inför ICC-analys för att se om valet av post-fixeringsmedel påverkar antikropparnas färgningsmönster (nukleärt, cytoplasmatiskt eller membranöst). CD45, CK5, CK7 och TTF-1 kunde valideras i studien. Det fanns inte någon signifikant skillnad (p > 0,05) i bedömningskategorierna (bakgrund, inmärkning och cellantal) mellan olika post-fixeringsmedel. Det fanns heller inga indikationer som visade att en särskild post-fixering gynnande ett särskilt färgningsmönster. Fler studier behövs för att förstå den preanalytiska fasens påverkan på ICC- analys av cytologiskt material. I samband med punktionscytologi skulle preparering enligt TP-metoden säkerställa tillgången på cellmaterial för ICC-analys. / Immunohistochemistry (IHC) and immunocytochemistry (ICC) are crucial tools in cancer diagnostics for establishing diagnosis. Both histological and cytological materials can be used for immunochemical analysis, but they require different preparations. The pre-analytical phase affects the epitope and antigen availability as well as immunoreactivity, making the choice of pre- and post-fixation and their impact on cytological material important to understand. ICC is usually applied to cytological material prepared using the cell block technique (CBT), but sometimes there is a lack of extracted material for analysis. Antibodies can differentiate tumor origins, subtypes, and inflammatory conditions, which is essential for clinical diagnostics and treatment decisions. In clinical pathology and cytology in Kalmar, a recurring issue is the lack of cell material for CBT preparation in fine-needle aspiration cytology. This study aimed to validate antibodies (calretinin, CD45, CK5, CK7, SOX-10, EpCAM, and TTF-1) on cytological material (n=10 pleural effusions) prepared using the ThinPrep™ method (TP method). The study also aimed to study the effects of post-fixation in 4% formalin, 96% ethanol, or without fixation medium before ICC analysis to see if the choice of post-fixation medium affects the staining patterns of the antibodies (nuclear, cytoplasmic, or membranous). CD45, CK5, CK7, and TTF-1 were validated in the study. There was no significant difference (p > 0.05) in the assessment categories (background, staining, and cell count) between different post-fixation media. There were also no indications that a particular post-fixation medium favored a specific staining pattern. Further studies are needed to understand the pre-analytical phase's impact on ICC analysis of cytological material. In fine-needle aspiration cytology, preparation using the TP method would ensure the availability of cell material for ICC analysis.

Submitted by Isaac Francisco de Souza Dias (isaac.souzadias@ufpe.br) on 2016-03-04T17:16:30Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MICHELINE OLIVEIRA LOBO PEREIRA DA COSTA- TESE DOUTORADO -23-10-2015.pdf: 15371820 bytes, checksum: 4afd4b560150641ba2dc59721bbb563e (MD5) / Made available in DSpace on 2016-03-04T17:16:30Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) MICHELINE OLIVEIRA LOBO PEREIRA DA COSTA- TESE DOUTORADO -23-10-2015.pdf: 15371820 bytes, checksum: 4afd4b560150641ba2dc59721bbb563e (MD5) Previous issue date: 2015-01-23 / CAPES / O presente estudo avaliou o desempenho da metodologia citológica de base líquida (ThinPrep-TP) com o da citologia convencional de Papanicolaou (CC) no diagnóstico de alterações citopatológicas e de resultados insatisfatórios, sob a visão de um serviço público no estado de Pernambuco (LACEN-PE) e da Fundação Oncocentro de São Paulo (FOSP-SP). Também foram avaliadas as associações entre a presença de coinfecções genitais dos subtipos de Human papillomavirus (HPV) com Chlamydia trachomatis (CT) e/ou outras microfloras, com os diferentes estágios de alterações cervicais das pacientes. A população do estudo foi de 525 mulheres na faixa etária dos 18-65 anos, atendidas por demanda espontânea, pelas Unidades Básicas de Saúde no Estado de Pernambuco, no período de abril a novembro de 2011. Um questionário padronizado com informações sobre características sociodemográficas, sexuais, reprodutivas e de hábitos (tabagismo, consumo de bebidas alcoólicas e uso de drogas) foram obtidos de todas as pacientes do estudo. A presença de DNA do HPV e da CT foram diagnósticadas através da reação em cadeia da polimerase (PCR) e o exame citológico foi realizado para detecção das demais infecções. Para avaliar a relação das infecções genitais com a presença de alterações intraepiteliais cervicais, foi utilizado o teste exato de Fisher. Os resultados mostraram que 11,05% das pacientes tinham menos de 25 anos, 30,86% eram solteiras, 6,86% tiveram mais de 5 parceiros sexuais, 44% não faziam uso de métodos contraceptivos, 38,85% eram usuárias de álcool, 24,38% eram fumantes e 3,24% haviam consumido drogas. Além disso, 42,01% tinham queixas ginecológicas; e 12,19% história pregressa de doenças sexualmente transmissíveis (DST). Quando comparadas a eficiência das técnicas TP e CC sob a visão do LACEN-PE, observou-se que as duas metodologias avaliadas apresentaram fraca concordância entre os métodos (k=0,19;95%IC(0,11-0,26); p<<0,001). A metodologia TP reduziu a taxa de resultados insatisfatórios de 4,38% para 1,71% (×2= 5,28; p=0,02), e o número de alterações citopatológicas diagnosticadas aumentaram de 2,47% para 3,04%. Porém sob a avaliação da FOSP-SP, as duas metodologias apresentaram concordância (k=0,39;95%IC(0,29-0,50); p<<0,001). A metodologia TP apresentou taxas insatisfatórias e praticamente semelhantes de 3,20% para 3,60% (×2= 5,00; p=0,17), e de alterações citopatológicas de 5,60% para 4,20%. Em 87 casos, foram observadas alterações cervicais por uma das metodologias utilizadas; destas, em 83,91% foram detectados CT e 82,76% foram positivas para HPV, ocorrendo coinfecção em 65 casos (74,7%). Em 93,1% dos casos houveram alterações colposcópicas, observando-se uma associação estatisticamente significativa entre coinfecção HPV-CT e presença de lesão (p=0.037). Outras microfloras encontradas foram Gardnerella vaginalis (35,6%), cocos (18,4%), Candida sp (9,2%), Trichomonas vaginalis (6,9%), Lactobacillus sp (4,6%) e herpesvírus (1,15%). Flora mista (coinfecção por várias microfloras – com exceção de CT simultaneamente) ocorreram em 41,38% dos casos. No entanto, nenhuma associação da presença desses microrganismos não-virais e os herpesvírus com a gravidade das lesões intraepiteliais foram encontradas. Os subtipos de HPV mais frequentes foram 16 e 31 (34,3% e 17,15%, respectivamente). Porém nas lesões de maior gravidade, os mais prevalentes foram 16 e 18. Desta forma, concluimos que este estudo demonstra a superioridade da metodologia TP no diagnóstico citológico das amostras cervicais o que poderá contribuir na diminuição de possíveis perdas por repetição citológica e seguimento das pacientes. É importante chamar atenção para as infecções genitais, em especial a CT, que devem sem investigadas e tratadas adequadamente, haja vista, que coinfecções com o HPV estão associadas ao favorecimento de lesões cervicais, e podem evoluir a graus mais avançados. / This study evaluated the performance of cytological methodology net basis (ThinPrep-TP) with the conventional Pap cytology (CC) in the diagnosis of cytopathological findings and unsatisfactory results under the vision of a public service in the state of Pernambuco (LACEN -PE) and Oncocentro Foundation of São Paulo (FOSP-SP). We also evaluated whether there is an association between the presence of genital co-infections of human papillomavirus subtypes (HPV) with Chlamydia trachomatis (CT) and or other microflora, with the different stages of cervical abnormalities of patients. The study population of 525 women between the ages of 18-65 years, assisted by spontaneous demand, the Basic Health Units in the State of Pernambuco, in the period from April to November 2011. A standardized questionnaire with information on sociodemographic characteristics, sexual, reproductive habits (such as smoking, alcohol consumption and drug use) were obtained from all study patients. The presence of HPV DNA and CT were both diagnosed by polymerase chain reaction (PCR) and cytological examination was performed to detect other infections. To evaluate the relationship of genital infections with the presence of cervical intraepithelial changes, Fisher's exact test was used. The results showed that 11.05% of patients were under 25yrs, 30.86% were single, 6.86% had more than five sexual partners, 44% did not use contraception, 38.85% were users of alcohol, 24.38% smokers and 3.24% had used drugs before. Moreover, gynecological complaints were 42.01%; and 12.19% history of STD. When comparing the efficiency of two techniques used in diagóstico of cervical abnormalities in the view of LACEN-PE, it was observed that the two methodologies evaluated showed poor agreement between the methods (k = 0.19; 95% CI (0,11- 0.26), p << 0.001). TP method reduced the rate of unsatisfactory 4.38% to 1.71% (5.28 = 2 × p = 0.02) and the number of cytopathological changes diagnosed increased 2.47% to 3 04%. But in the assessment of FOSP-SP, the two methodologies showed reasonable agreement (k = 0.39; 95% CI (0.29 to 0.50); p << 0.001) .The TP methodology showed unsatisfactory results virtually rates similar 3.20% to 3.60% (× 2 = 5.00; p = 0.17), and cytological changes from 5.60% to 4.20%. In 87 cases, cervical abnormalities were observed by any of the methods used, and of these, 83.91% were detected CT, and 82.76% were positive for HPV, occurring co-infection in 65 cases (74.7%). In 93.1% of cases there were colposcopic changes, observing a statistically significant association between co-infection HPV-CT and presence of lesions (p = 0.037). Other microflora were found Gardnerella vaginalis (35.6%), coconut (18.4%), Candida sp (9.2%), Trichomonas vaginalis (6.9%), Lactobacillus sp (4.6%) and herpesviruses (1.15%). Mixed flora (co-infection with various microflora - CT exception with both) occurred in 41.38% of cases. However, no association between the presence of non-viral organisms and herpesviruses with the severity of intraepithelial lesions were found. The most common HPV subtypes there were 16 and 31 (34.3% and 17.15%, respectively). But in more severe injuries, the most prevalent were 16 and 18. Thus, we conclude that this study demonstrates the superiority of TP methodology in the cytological diagnosis of cervical samples which could contribute to decrease possible losses by repetition and cytologic follow-up of patients . It is important to draw attention to the genital infections, especially CT, which are not investigated and dealt with appropriately, given that co-infections with HPV are associated with the favoring of cervical lesions, and can progress to more advanced degrees.