Sysmex XE-5000 är en automatiserad cellräknare som utför mätningar enligt olikamätprinciper, de två som tillämpats i projektet är RF/DC(Radio Frequency/DirectCurrent) samt Flödescytometri med halvledarlaser. RF/DC bygger på förändringari radiofrekventa resistansen och likspänningsresistansen. Förändringar i denradiofrekventa resistansen (RF) ger information om densiteten i cellernas inre(exempelvis kärnans storlek) och förändringar i likspänningsresistensen (DC) gerinformation om blodcellernas storlek. Flödescytometri definierar ett mått påcellers fysiologiska och kemiska egenskaper. Detektion av cellerna sker genom attde bestrålas med en laserstråle samtidigt som de passerar en och en i instrumentet.Informationen som fås ut från flödescytometri inkluderar spritt ljus ochfluorescens. Sysmex XE-5000 arbetar med flera olika felmeddelanden, så kalladelarm. Ett eller flera larm indikerar att det finns en ökad risk för förekomst avabnormala celler och kan enbart uteslutas genom en manuell differentialräkning. Istudien har tre larm, vilka indikerar närvaron av onormala leukocyter, undersökts:”Blasts?”, ”Atypical Lympho?” och ”Abn Lympho/L_Blasts?”. Syftet medprojektet är att jämföra nuvarande beräkningar med en ny mjukvara (eMM) förlarmen och utvärdera om de ger ett mindre antal falskt positiva larm frånhematologiinstrumentet Sysmex XE-5000. Prover med något av ovanstående larmvaldes ut och analyserades först med nuvarande inställningar på instrumentet ochdärefter med de nya beräkningarna för eMM. Resultatet visar på att antalet falsktpositiva prover minskar och även att antalet dubblettlarm minskar. / Sysmex XE-5000 is an automated cell counter that performs measurements withdifferent principles. The two applied in this project are RF/DC (RadioFrequency/Direct Current) and Flow cytometry with semiconductor laser. RF/DCis based on changes in radio frequency resistance and direct current voltage.Changes in RF provide information about the density of the cell’s internalstructure (e.g. the nucleus) and changes in DC provide information about the sizeof the blood cells. Flow Cytometry define as physiological and chemicalproperties of the cell. Detection of cells is achieved by the irradiation with a laserbeam while passing through one by one. The information obtained from flowcytometry includes scattered light and fluorescence. Sysmex XE-5000 works withseveral different error messages, so-called alarm. One or more alarm indicates thatthere is an increased risk for the presence of abnormal cells and this can only beruled out by a manual differential count. In this study three alarms, which indicatethe presence of abnormal white blood cells, were analyzed: “Blasts?”, AtypicalLympho?” and “Abn Lympho/L_Blasts?”. The project aims to compare thecurrent calculations with the new software (eMM) for the alarms and evaluate ifthey provide a smaller number of false positive alarms from the hematologyinstrument Sysmex XE-5000. Samples with one or two of the alarms wereselected and analyzed with the current settings and then with the new settings foreMM. The result showed that the number of false-positive samples was reducedand that the number of duplicate alarms decreased.

Este estudo teve o objetivo de avaliar a eficácia da movimentação precoce do braço, no controle da formação de seroma, em mulheres com câncer de mama submetidas a linfadenectomia axilar (LA). Foi realizado um estudo comparativo com a participação de 39 mulheres, com diagnóstico de câncer de mama, unilateral de qualquer extensão, submetidas à cirurgia com LA. A coleta dos dados foi realizada de julho de 2004 à janeiro de 2005, Setor de Onco-ginecologia e Mastologia do Departamento de Ginecologia e Obstetrícia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP). As mulheres foram divididas, aleatoriamente, em dois grupos: Grupo Movimento (GM) e Grupo Controle (GC). No GC, as 20 mulheres receberam as orientações rotineiras da unidade e no GM, as 19 mulheres foram orientadas à movimentação precoce e sistemática do braço, com um programa específico de movimentos iniciados antes da alta hospitalar e continuando no domicílio. Todas as participantes foram avaliadas antes da cirurgia, no primeiro dia pós operatório, de 5 a 10 dias e de 30 a 40 dia depois da cirurgia. Através do teste estatístico de Mann-Whitney observou-se que os grupos foram homogêneos para as variáveis idade e índice de massa corpórea, apesar de o número de sujeitos não ter sido estatisticamente significante. O tempo de permanência do dreno e o volume drenado, foram menores no GM. A incidência de seroma foi igual para os dois grupos, mas a resolução foi mais rápida e menos invasiva no GM. Pode-se assim concluir que apesar de a amostra não ter sido estatisticamente significante, a movimentação precoce e sistematizada favoreceu uma melhor absorção do seroma e não esteve associada a outras complicações cirúrgicas. / This study aimed to evaluate the efficacy of early arm movement in the control of seroma formation, in women who underwent complete axillary lymphadenectomy (AL) due to breast cancer. A comparative study involved 39 women with diagnosis of unilateral breast cancer of any extension who underwent LA. Data were collected between July 2004 and January 2005, at the onco-gynecology and mastology sector - Gynecology and Obstetric Department of the University of São Paulo at Ribeirão Preto Medical School Hospital das Clínicas. Participants were randomly distributed in two groups: Movement Group (MG) and Control Group (CG). In the CG, 20 women received routine orientation while, in the MG, 19 women were oriented towards systematic early arm movement, with a specific movement program initiated before hospital discharge and continued at home. All women were evaluated the day before surgery, the first day after surgery, 5-10 days and 30-40 days after surgery. The Mann-Whitney statistical test revealed that the groups were homogeneous in terms of age and body mass index, although the number of subjects was found statistically insignificant. The permanence of the drain and the volume drained were lower in MG. Incidence levels of seroma were equal in the two groups, but the resolution was faster and less invasive in MG. In spite of a statistically insignificant number of subjects, early and systematic arm movement provided better seroma absorption and was not associated with other surgical complications.

In der vorliegenden Arbeit wurden 15 gesunde Probanden und vier Patienten mit dem primären humoralen Immundefekt "Common Variable Immunodeficiency" (CVID) auf den Immunphänotyp ihrer Lymphozyten sowie die Expression des Transkriptionsfaktors PRDI-BF1/Blimp-1 hin untersucht. Zu Kontrollzwecken wurden zusätzlich einige permanente Lymphozyten-Zelllinien verwendet (B-lymphoblastoide Zelllinien und Jurkat-Zellen). Nach der Blutentnahme wurde zunächst in Vollblut-Lyse-Technik der Immunphänotyp bestimmt, begleitend wurde ein großes Blutbild auf einem Hämatologieautomaten gemessen. Im Vergleich zu den gesunden Probanden zeigten die CVID-Patienten im Differentialblutbild folgende statistisch signifikante Abweichungen: relative Lymphopenie, relative Neutrophilie, relative und absolute Eosinopenie (insgesamt im Sinne einer entzündlichen Reaktion). Durchflusszytometrisch zeigten die Patienten folgende Auffälligkeiten: relative Expansion der zytotoxischen T-Lymphozyten, absolute Verminderung der NK-Zellen, relative Expansion HLA-DR-positiver Lymphozyten, absolute Verminderung CD27-positiver Lymphozyten, relative und absolute Verminderung CD27-positiver B-Zellen (B-Gedächtnis-Zellen). Bei allen vier Patienten war der Anteil IgM-negativer ("geswitchter") Memory-Zellen an den gesamten B-Lymphozyten stark vermindert. Nach Stimulation mit Staphylococcus aureus Stamm Cowan I und Interleukin 2 waren bei allen untersuchten Patienten und gesunden Probanden Immunglobuline im Zellkulturüberstand sowie PRDI-BF1-mRNA im Zelllysat nachweisbar, sodass auf einen gravierenden Defekt von PRDI-BF1 als Ursache der CVID (z. B. im Sinne einer homozygoten Gendeletion) kein Hinweis bestand. Bei den als Negativkontrolle vorgesehenen T-Lymphozyten und in einer permanenten T-Zelllinie (Jurkat) wurde überraschend ebenfalls PRDI-BF1-mRNA nachgewiesen. Dieser Befund konnte durch mehrfache Wiederholung, Kontrollen und hohe Aufreinigung der T-Lymphozyten sowie Auftrennung in ihre Subpopulationen bestätigt werden und wurde von anderen Arbeitsgruppen ebenfalls reproduziert und publiziert. Die Ergebnisse ließen vermuten, dass PRDI-BF1 bei Antigen-erfahrenen, CD45RA-negativen T-Lymphozyten stärker exprimiert wird und daher ebenso wie bei B-Lymphozyten eine Rolle in deren terminaler Differenzierung spielt. / In this paper 15 healthy subjects and four patients with common variable immunodeficiency (CVID, a primary humoral immunodeficiency) were studied regarding the immune phenotype of their lymphocytes and the expression of the transcription factor PRDI-BF1/Blimp-1. For control purposes, some additional permanent lymphocyte cell lines were used (B-lymphoblastoid cell lines and Jurkat cells). After blood was drawn, the immune phenotype was determined using whole blood lysis technique. Additionally a complete blood count was measured on a hematology analyser. Compared to healthy subjects the CVID patients showed the following statistically significant differences in the differential blood count: relative lymphopenia, relative neutrophilia, relative and absolute eosinopenia (overall in terms of an inflammatory reaction). Flow cytometry showed the following abnormalities: relative expansion of cytotoxic T lymphocytes, absolute reduction of NK cells, relative expansion of HLA-DR positive lymphocytes, absolute reduction of CD27 positive lymphocytes, relative and absolute decrease in CD27 positive B cells (memory B cells). In all four patients the proportion of IgM negative (switched) memory cells to total B lymphocytes was greatly reduced. After stimulation with Staphylococcus aureus strain Cowan I and interleukin 2 in all studied patients and healthy subjects immunoglobulins in the cell culture supernatant and PRDI-BF1 mRNA in the cell lysate were detected, so that there was no evidence of a serious defect of PRDI-BF1 as a cause of CVID (e. g. in terms of a homozygous gene deletion). In the T lymphocytes and in a permanent T cell line (Jurkat) intended as negative controls PRDI-BF1 mRNA was surprisingly detected as well. This finding was confirmed through multiple repetitions, controls and high purification and separation of T lymphocytes in their subpopulations and was also reproduced and published by other research groups. The results suggested that PRDI-BF1 is expressed at higher levels in antigen experienced, CD45RA negative T lymphocytes and, therefore, plays a role in their terminal differentiation.

Este estudo teve o objetivo de avaliar a eficácia da movimentação precoce do braço, no controle da formação de seroma, em mulheres com câncer de mama submetidas a linfadenectomia axilar (LA). Foi realizado um estudo comparativo com a participação de 39 mulheres, com diagnóstico de câncer de mama, unilateral de qualquer extensão, submetidas à cirurgia com LA. A coleta dos dados foi realizada de julho de 2004 à janeiro de 2005, Setor de Onco-ginecologia e Mastologia do Departamento de Ginecologia e Obstetrícia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FMRP-USP). As mulheres foram divididas, aleatoriamente, em dois grupos: Grupo Movimento (GM) e Grupo Controle (GC). No GC, as 20 mulheres receberam as orientações rotineiras da unidade e no GM, as 19 mulheres foram orientadas à movimentação precoce e sistemática do braço, com um programa específico de movimentos iniciados antes da alta hospitalar e continuando no domicílio. Todas as participantes foram avaliadas antes da cirurgia, no primeiro dia pós operatório, de 5 a 10 dias e de 30 a 40 dia depois da cirurgia. Através do teste estatístico de Mann-Whitney observou-se que os grupos foram homogêneos para as variáveis idade e índice de massa corpórea, apesar de o número de sujeitos não ter sido estatisticamente significante. O tempo de permanência do dreno e o volume drenado, foram menores no GM. A incidência de seroma foi igual para os dois grupos, mas a resolução foi mais rápida e menos invasiva no GM. Pode-se assim concluir que apesar de a amostra não ter sido estatisticamente significante, a movimentação precoce e sistematizada favoreceu uma melhor absorção do seroma e não esteve associada a outras complicações cirúrgicas. / This study aimed to evaluate the efficacy of early arm movement in the control of seroma formation, in women who underwent complete axillary lymphadenectomy (AL) due to breast cancer. A comparative study involved 39 women with diagnosis of unilateral breast cancer of any extension who underwent LA. Data were collected between July 2004 and January 2005, at the onco-gynecology and mastology sector - Gynecology and Obstetric Department of the University of São Paulo at Ribeirão Preto Medical School Hospital das Clínicas. Participants were randomly distributed in two groups: Movement Group (MG) and Control Group (CG). In the CG, 20 women received routine orientation while, in the MG, 19 women were oriented towards systematic early arm movement, with a specific movement program initiated before hospital discharge and continued at home. All women were evaluated the day before surgery, the first day after surgery, 5-10 days and 30-40 days after surgery. The Mann-Whitney statistical test revealed that the groups were homogeneous in terms of age and body mass index, although the number of subjects was found statistically insignificant. The permanence of the drain and the volume drained were lower in MG. Incidence levels of seroma were equal in the two groups, but the resolution was faster and less invasive in MG. In spite of a statistically insignificant number of subjects, early and systematic arm movement provided better seroma absorption and was not associated with other surgical complications.

In situ follicular neoplasia (ISFN) is defined as a monoclonal proliferation of B cells with immunophenotypic and genetic features of follicular lymphoma (FL) but confined to germinal centers of lymph nodes or other organs. It may not be associated with underlying overt lymphoma. It can be associated with lymphoproliferative disorders other than FL. A fifty-seven-year-old caucasian male initially presented with atypical chest pain, which led to cardiology evaluation. Patient underwent a coronary CT angiogram, which revealed a calcium score of 0, however also incidentally revealed mediastinal lymphadenopathy. Patient had a bronchoscopy which revealed no endobronchial lesions bilaterally. Using endo-bronchial ultrasound, right carinal lymph node was visualized, and trans-bronchial fine needle aspiration was performed. Cytology was positive for necrotic lesion with atypical cells. Patient had a dedicated CT scan of chest which showed enlarged sub-carinal lymph node measuring 3.3 x 3.0 cm. PET/CT scan showed increased uptake in the sub-carinal lymph nodes, also increased uptake of mid para-esophageal lymph nodes. It also showed some low-grade lymphadenopathy in right lower paratracheal region as well as mesenteric lymphadenopathy with misty appearance. Small pulmonary nodules were also noted in right middle and lower lobes with no associated uptake. Patient was scheduled for a mediastinoscopy and lymph node dissection. Patient proceeded with mediastinoscopy and a total of 4 lymph node specimens were removed from level 4R and level 7. Pathology from one of the lymph nodes revealed necrotizing granulomatous inflammation with staining consistent with histoplasmosis. Interestingly, two other lymph nodes showed in situ follicular neoplasia. Immunohistochemical stains demonstrated rare secondary lymphoid follicles with unremarkable morphology, showing strong germinal center staining with BCL2. FISH analysis was normal indicating absence of t(14;18). Pathology showed morphologically unremarkable B-cell nodules, concentrated in the cortical area which were CD20 positive and BCL2-positive. Patient underwent subsequent treatment with anti-fungal agents for the Histoplasmosis and is currently under surveillance for in-situ follicular lymphoma. In-situ follicular neoplasia is considered a premalignant lesion and a precursor of follicular lymphoma. Incidence of ISFN is difficult to ascertain, as it is usually a subclinical diagnosis. Incidence of FL is 3.18 per 100,000 population in the United States and findings suggest that ISFN is likely more frequent than that. Also, similar to FL, ISFN is seen in middle-aged and older individuals, mean age being around the fifth decade of life.Incidentally found ISFN without prior or simultaneous lymphoma is associated with a very low rate of clinical progression. Because some cases of ISFN are associated with prior or concurrent lymphoma, screening studies including computed tomography (CT) scan and bone marrow biopsy should be conducted after the diagnosis of ISFN is made. In the absence of overt lymphoma, it has been recommended that patients with ISFN be observed without chemotherapy, based on the very low incidence of progression into overt FL. The clinical significance of ISFN is not fully understood, however studies have demonstrated that incidentally found ISFN without prior or simultaneous lymphoma is associated with a very low rate of clinical progression. (

A síndrome de emagrecimento progressivo (SEP) é responsável por elevada morbidade e mortalidade de calitriquídeos mantidos em cativeiro em diferentes instituições. Essa síndrome representa um desafio aos médicos veterinários por suas características ainda pouco esclarecidas e são poucos os estudos multidisciplinares que visam à avaliação dos diferentes sistemas, como os órgãos linfo-hematopoiéticos. O objetivo foi caracterizar a evolução e duração da SEP, associando os dados clínicos, laboratoriais e anatomopatológicos dos órgãos linfo-hematopoiéticos de saguis naturalmente acometidos por SEP no cativeiro. Foram analisadas as fichas clínicas, necroscópicas e os resultados das amostras de sangue e urina de 47 saguis doentes, Callithrix spp., machos e fêmeas, que foram a óbito devido a SEP e eram provenientes de dois criadouros diferentes do estado de São Paulo. Os fragmentos dos órgãos linfo-hematopoiéticos, intestino delgado e do fígado foram processados e avaliados. Os resultados caracterizaram que a SEP acomete calitriquídeos de espécies diferentes, adultos, sem predisposição sexual, mantidos sob condições estáveis de manejo por em média 42 meses e a duração clínica varia de 41 dias a 1 ano e 7 meses. As características clínicas na fase inicial foram predominantemente sinais gastrintestinais e na fase terminal, sinais gastrintestinais e extra-intestinais. A anemia macrocítica normo ou hipocrômica com policromasia, esferocitose, presença de corpúsculos de Heinz e hemoglobinúria foi a alteração hematológica mais frequente. As lesões dos órgãos linfo-hematopoiéticos foram características de anemia hemolítica ou foram inespecíficas e reacionais caracterizadas por hiperplasia ou depleção das células da medula óssea, baço e linfonodo e lesões degenerativas no fígado. Na SEP, a associação clínica, laboratorial e anatomopatológica possibilitou a caracterização da evolução e duração clínica, da anemia e das alterações dos órgãos linfo-hematopoiéticos, cujas lesões foram consideradas secundárias à desnutrição crônica e progressiva decorrente da severa enterite atrófica. / Wasting marmoset syndrome (WMS) causes high morbidity and mortality of marmosets and tamarins kept in captivity in different colonies. WMS challenges the veterinarian due to its unclear and not established features and there are few multidisciplinary studies that carried out an evaluation of different systems, such the lymphohematopoietic system. The aim was described the duration and evolution of illness based on an association of clinical, laboratory and pathological aspects of WMS. Medical record, laboratory data and pathological findings were analyzed of 47 Callithrix spp., males and females, sick marmosets that died due to WMS in two different colonies in São Paulo state. Tissue samples of small intestine, lymphohematopoietc system and liver were histological processed and evaluated. The results showed that WMS affects adult marmosets of different species; there are no sex-related differences, and the marmosets are at least 42 months under similar general management at colony. The clinical duration of WMS is from 41 days to 1 year and 7 months. The clinical features were gastrointestinal symptoms in the beginning and extra-gastrointestinal and gastrointestinal signs in the end. Normochromic or hypochromic macrocytic anemia with polychromasia, spherocytes, Heinz bodies, and hemoglobinuria is the common hematological result. The lymphohematopoietic system lesions were the common findings of hemolytic anemia or unspecific and reacting features such as hyperplasia or depletion of cell numbers of bone marrow, spleen and lymph node, and degenerative lesions of liver. The clinical, laboratory and pathological association allowed the characterization of evolution and duration of the WMS, the anemia and the lesions of lymphohematopoietic organs which lesions were considered secondary to chronic and progressive malnutrition as a result of severe atrophic.

A síndrome de emagrecimento progressivo (SEP) é responsável por elevada morbidade e mortalidade de calitriquídeos mantidos em cativeiro em diferentes instituições. Essa síndrome representa um desafio aos médicos veterinários por suas características ainda pouco esclarecidas e são poucos os estudos multidisciplinares que visam à avaliação dos diferentes sistemas, como os órgãos linfo-hematopoiéticos. O objetivo foi caracterizar a evolução e duração da SEP, associando os dados clínicos, laboratoriais e anatomopatológicos dos órgãos linfo-hematopoiéticos de saguis naturalmente acometidos por SEP no cativeiro. Foram analisadas as fichas clínicas, necroscópicas e os resultados das amostras de sangue e urina de 47 saguis doentes, Callithrix spp., machos e fêmeas, que foram a óbito devido a SEP e eram provenientes de dois criadouros diferentes do estado de São Paulo. Os fragmentos dos órgãos linfo-hematopoiéticos, intestino delgado e do fígado foram processados e avaliados. Os resultados caracterizaram que a SEP acomete calitriquídeos de espécies diferentes, adultos, sem predisposição sexual, mantidos sob condições estáveis de manejo por em média 42 meses e a duração clínica varia de 41 dias a 1 ano e 7 meses. As características clínicas na fase inicial foram predominantemente sinais gastrintestinais e na fase terminal, sinais gastrintestinais e extra-intestinais. A anemia macrocítica normo ou hipocrômica com policromasia, esferocitose, presença de corpúsculos de Heinz e hemoglobinúria foi a alteração hematológica mais frequente. As lesões dos órgãos linfo-hematopoiéticos foram características de anemia hemolítica ou foram inespecíficas e reacionais caracterizadas por hiperplasia ou depleção das células da medula óssea, baço e linfonodo e lesões degenerativas no fígado. Na SEP, a associação clínica, laboratorial e anatomopatológica possibilitou a caracterização da evolução e duração clínica, da anemia e das alterações dos órgãos linfo-hematopoiéticos, cujas lesões foram consideradas secundárias à desnutrição crônica e progressiva decorrente da severa enterite atrófica. / Wasting marmoset syndrome (WMS) causes high morbidity and mortality of marmosets and tamarins kept in captivity in different colonies. WMS challenges the veterinarian due to its unclear and not established features and there are few multidisciplinary studies that carried out an evaluation of different systems, such the lymphohematopoietic system. The aim was described the duration and evolution of illness based on an association of clinical, laboratory and pathological aspects of WMS. Medical record, laboratory data and pathological findings were analyzed of 47 Callithrix spp., males and females, sick marmosets that died due to WMS in two different colonies in São Paulo state. Tissue samples of small intestine, lymphohematopoietc system and liver were histological processed and evaluated. The results showed that WMS affects adult marmosets of different species; there are no sex-related differences, and the marmosets are at least 42 months under similar general management at colony. The clinical duration of WMS is from 41 days to 1 year and 7 months. The clinical features were gastrointestinal symptoms in the beginning and extra-gastrointestinal and gastrointestinal signs in the end. Normochromic or hypochromic macrocytic anemia with polychromasia, spherocytes, Heinz bodies, and hemoglobinuria is the common hematological result. The lymphohematopoietic system lesions were the common findings of hemolytic anemia or unspecific and reacting features such as hyperplasia or depletion of cell numbers of bone marrow, spleen and lymph node, and degenerative lesions of liver. The clinical, laboratory and pathological association allowed the characterization of evolution and duration of the WMS, the anemia and the lesions of lymphohematopoietic organs which lesions were considered secondary to chronic and progressive malnutrition as a result of severe atrophic.

Epidermal homeostasis, including proper turnover of keratinocytes, plays important role in the barrier function and serine proteases and their inhibitors are the key players. Activated proteases cleave desmosomes in uppermost layer and thus shed the cells from the epidermal surface. Therefore the serine protease inhibitors are secreted in lower epidermal layers to prevent premature activation of proteases and consequent disruption of epidermal barrier. The most studied inhibitors in epidermis belong to Serine proteases inhibitors Kazal-type family (SPINK). This diploma thesis is aimed to investigate function of murine SPINK6 in epidermal compartment in vivo. To achieve this, the transgenic mice overexpressing mSPINK6 under modified human involucrin promoter was generated. Two of five transgenic lines exhibited higher expression of mSPINK6 at mRNA and protein levels. The mSPINK6 transgenic mice are viable with no apparent phenotype. The small but in most cases not significant differences were observed on microscopic level among mSPINK6 transgenic and wild type animals In conclusion, this work showed that mSPINK6 does not play major role in skin homeostasis but gains significant importance under specific challenges of epidermal barrier. Therefore mSPINK6 transgenic mice, in combination with other deletion or...