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Master's Dissertation
DOI
https://doi.org/10.11606/D.10.2016.tde-04052016-154705
Document
Author
Full name
Fábio Novelli Martorelli
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2016
Supervisor
Committee
Hagiwara, Mitika Kuribayashi (President)
Gomes, Simone Gonçalves Rodrigues
Oliveira, Clair Motos de
Title in Portuguese
Caracterização da anemia em cadelas com piometra
Keywords in Portuguese
Anemia hemolítica imunomediada
Cadelas
Citometria de fluxo
Piometra
Abstract in Portuguese
A piometra é uma condição mórbida caracterizada pela inflamação do útero com acúmulo de exsudatos, resultante de ações hormonais e geralmente associada à presença de bactérias no lúmen uterino. A anemia é a alteração hematológica mais frequentemente observada em cadelas com piometra e está associada à cronicidade da doença, diminuição da eritropoiese, devido ao efeito toxêmico na medula óssea, diminuição da disponibilidade de ferro ou perda de sangue para o útero. Adicionalmente, o efeito das toxinas bacterianas e os radicais livres gerados pelo metabolismo oxidativo dos neutrófilos podem resultar na modificação da estrutura antigênica da membrana do eritrócito, permitindo a ligação de imunoglobulinas em sua superfície e acelerando a destruição eritrocitária. Essa hipótese pode ser comprovada pela detecção de imunocomplexos na superfície eritrocitária de cadelas com piometra. O diagnóstico de piometra foi estabelecido em 33 cadelas atendidas no Serviço de Obstetrícia/Ginecologia do Hospital Veterinário da Universidade de São Paulo com base na anamnese, exame físico e exames subsidiários (ultrassonografia, hemograma e concentrações séricas de ureia e creatinina). As amostras sanguíneas foram coletadas em dois momentos. A primeira anterior a ovariosalpingohisterectomia (OSH) e a segunda, sete a dez dias após a OSH. A quantificação de hemácias com deposição de imunocomplexos IgG e IgM foi realizada utilizando-se anticorpos anti-IgG e anti-IgM (Bethyl®Laboratories) conjugadas a fluoresceína de isotiocianato (FITC), e a leitura realizada com citômetro de fluxo (FACS Calibur; Becton, Dickinson and Company© 2007 BD), sendo os resultados expressos em percentual de hemácias marcadas. Foram utilizados o Teste de Shapiro-Wilk para a avaliação da distribuição de dados e a comparação entre os grupos controle, pré e pós-OSH foi realizada valendo-se do Teste t ou Teste t pareado e Correlação de Pearson, e do Teste U de Mann-Whitney e Correlação de Spearman, para as variáveis com distribuição normal e não-normal, respectivamente. O valor de alfa estipulado foi de 0,05. Analisando os valores hematológicos de cada um dos cães incluídos no estudo, observa-se que 19 (57,6%) apresentavam anemia normocítica normocrômica não regenerativa no momento pré-OSH e cinco (15,2%) no momento pós-OSH. Em cães do grupo controle foram observadas 0,14 - 0,77% (0,43±0,18%) de hemácias marcadas com anticorpos anti-IgG FITC e 0,29 - 9,58% (0,68±0,29%) para anticorpos anti-IgM FITC. Já nos cães com piometra, foram encontradas 0,14 - 4,19% (0,96±0,86%) de hemácias marcadas com anticorpos anti-IgG FITC e 0,29 - 9,58% (1,37±1,71%) com anticorpos anti-IgM FITC, antecedendo a OSH. No momento pós-OSH observou-se 0,18 - 16,2% (2,77±3,67%) de hemácias marcadas para anticorpos anti-IgG FITC e 0,15 - 19,8% (4,01±4,46%) para anticorpos anti-IgM FITC. O percentual de hemácias marcadas com anticorpos anti-IgG FITC diferiu entre os grupos controle e piometra, pré-OSH (p<0,001) e pós-OSH (p<0,001). Em relação a anticorpos anti-IgM FITC, não foram observadas diferenças entre os grupos controle e pré-OSH (p=0,09), porém, após a OSH houve aumento na marcação de hemácias, quando comparado ao grupo controle (p<0,001). Apenas alguns animais apresentaram mais de 5% de hemácias marcadas, e isto ocorreu, principalmente, no momento pós-OSH. Entretanto, não resultou no agravamento da anemia, indicando que a piometra em cadelas está associada à deposição de imunoglobulinas G ou M na superfície das hemácias, sem, no entanto, promover hemólise ou agravamento da anemia
Title in English
Characterization of anemia in bitches with pyometra
Keywords in English
Bitches
Flow cytometry
Immune mediated hemolytic anemia
Pyometra
Abstract in English
The pyometra is a morbid condition characterized by inflammation of the uterus with exudates accumulation resulting from hormonal action and usually associated with the presence of bacteria in the uterine lumen. Anemia is the hematologic changes most frequently observed in bitches with pyometra and is associated with chronic disease, diminished erythropoiesis due to toxemic effect on the bone marrow, decreased iron availability or loss of blood to the uterus. Additionally, the effect of bacterial toxins and free radicals generated by neutrophil oxidative metabolism may result in the modification of antigenic structure of the erythrocyte membrane, allowing the binding of immunoglobulins on their surface and accelerating erythrocyte destruction. This hypothesis can be confirmed by detection of immune complexes in the erythrocyte surface dogs with pyometra. The diagnosis of pyometra was established in 33 dogs attended by the Obstetrics Service/Gynecology of Veterinary Hospital of the University of São Paulo based on history, physical examination and additional tests (ultrasound, blood count and serum concentrations of urea and creatinine). Blood samples were collected in two stages. The first prior to ovariohysterectomy (OSH) and the second, seven to ten days after OSH. Quantification of erythrocyte with deposition of IgG and IgM immune complexes was performed using antibodies anti-IgG and anti-IgM (Bethyl® Laboratories) conjugated to fluorescein isothiocyanate (FITC), and the reading performed with flow cytometry (FACS Calibur; Becton, Dickinson and Company© 2007 BD), and the results expressed as a percentage of red blood cells marked. The Shapiro-Wilk test was used to assess the distribution of data and the comparison between the control group, pre- and post-OSH was carried out making use of the t test or paired t test and Pearson correlation, and test U Mann-Whitney and Spearman correlation for the variables with normal and non-normal distribution, respectively. The stipulated alpha value was 0.05. The analysis of the hematological values of each of the dogs enrolled in the study, it was observed that 19 (57.6%) with anemia normocytic normochromic non-regenerative in the pre-OSH moment and 5 (15.2%) in the post-OSH moment. In control group of dogs were observed from 0.14 to 0.77% (0.43± 0.18%) of red blood cells marked with antibodies anti-IgG FITC and 0.29 to 9.58% (0.68± 0.29%) for antibodies anti-IgM FITC. Already in dogs with pyometra were found 0.14 to 4.19% (0.96 ± 0.86%) of red blood cells marked with antibodies anti-IgG FITC and 0.29 to 9.58% (1.37 ± 1.71%) with antibodies anti-IgM FITC, prior to OSH. In the post-OSH time it was observed 0.18 to 16.2% (2.77 ± 3.67%) of red blood cells marked for antibodies anti-IgG FITC and 0.15 to 19.8% (4.01 ± 4.46%) for antibodies anti-IgM FITC. The percentage of red blood cells marked with antibodies anti-IgG FITC differ between groups control and pyometra, pre-OSH (p<0.001) and post-OSH (p<0.001). Regarding the antibodies anti-IgM FITC, no differences were observed between the control and pre-OSH (p=0.09), however, after the OSH was no increase in labeling of red blood cells compared to the control group (p<0.001). Only a few animals have more than 5% of marked red blood cells, and this was mainly in the post-OSH time. However, not result in the worsening of anemia, indicating that pyometra in dogs is related to the deposition of immunoglobulin G or M on the surface of erythrocytes, without, however, promote hemolysis or worsening of anemia
 
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Publishing Date
2016-05-31
 
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