Assessment transcallosal Diaschisis in a model of focal cerebral ischemia in rats / Evaluación de la diásquisis transcallosa en un modelo de isquemia cerebral focal en ratas

Objective: To evaluate transcallosal changes after a local ischemic injury in rats by using the monoclonal marker anti-NeuN (Mouse anti-neuronal nuclei). Methods: Twenty eight adult, male, Wistar rats were subjected to focal injury in the right hemisphere. The technique used was the experimental model of focal ischemic injury through intraluminal suture of the middle cerebral artery. Analyses were made for the five groups: and after the lesion (control), at 24 h, 96 h, 10 days and 20 days. Exofocal neuronal damage was inferred from neuronal immunoreactivity changes to NeuN. Results: In the cortex contralateral to the lesion, immunoreactivity was diminished. This was most notable in the supragranular layers 24 h post ischemia. After 96 h, there was a generalized diminishment of the inmmunoreactivity in supra and infragranular layers. At 10 and 20 days, the tissue recovered some NeuN immunoreactivity, but there were set changes in the VI layer. Conclusion: The immunoreactive changes to NeuN support the process of interhemispheric diaschisis. Changes in immunoreactivity could indicate metabolic stress secondary to the disruption in connectivity to the site of lesion.

Objetivo: Evaluar los cambios exofocales transcallosos después de lesión isquémica focal en ratas, mediante marcación inmunohistoquímica con el anticuerpo monoclonal anti-NeuN (Mouse Anti-Neuronal Nuclei). Métodos: Se intervinieron 28 ratas machos Wistar adultas. Mediante el modelo experimental de isquemia cerebral focal del territorio de la arteria cerebral media por filamento intraluminal, se les ocasionó una lesión focal en el hemisferio derecho. Posteriormente se evaluó el hemisferio contralateral, marcando la población neuronal con el anticuerpo monoclonal anti-NeuN. Se definieron cinco grupos de evaluación: uno de control, 24 h, 96 h, 10 días y 20 días. Se evaluaron los cambios neuronales exofocales después de la lesión con base en la observación de los cambios en la inmunoreactividad de las neuronas al NeuN. Resultados: Se redujo la inmunoreactividad en la corteza contralateral a la lesión. Este fenómeno fue más notable en las capas supragranulares después de 24 h post isquemia. Después de 96 h hubo una disminución generalizada de la inmmunoreactivity en las capas supra e infragranulares. A los 10 y 20 días, el tejido recobró alguna inmunoreactividad NeuN, estos cambios se dieron en la capa VI. Conclusiones: Los cambios inmunorreactivos a NeuN apoyan el proceso de diasquisis interhemisférica. Los cambios en la inmunorreactividad podrían indicar estrés metabólico secundario a la interrupción en la conectividad con el sitio de la lesión.

A Hemolytic Transfusion Reaction due to Anti-Ku Antibody in a Patient with Knull Phenotype: The First Case in Korea / 대한진단검사의학회지

Knull phenotype completely lacks all Kell system antigens. Anti-Ku antibody is seen in immunized persons with Knull phenotype by transfusion or pregnancy. It can cause a fatal hemolytic transfusion reaction. A 66-yr-old male patient with liver cirrhosis visited emergency center due to acute bleeding. The patient was at hypovolemic shock status: his blood pressure was 80/50 mmHg, pulse rate was 110/min and hemoglobin level was 4.4 g/dL. Because of the presence of antibody against high incidence antigen, we could not find any compatible blood for the patient. Nevertheless, 4 units of packed RBCs had to be transfused. Moderate hemolytic transfusion reaction was developed after transfusion. At endoscopic examination, blood was spurting from gastric cardiac varix. Endoscopic histoacryl injection was tried, and bleeding was successfully controlled. After bleeding stopped, he was managed for anemia using steroid and other medical therapy instead of transfusion. His hemoglobin level was improved to 7.7 g/dL at the time of discharge. Later he has been proved to have a Knull phenotype, which is very rare, and anti-Ku antibody. This report is the first case of anti-Ku in a Knull phenotype person in Korea, who experienced a moderate hemolytic transfusion reaction.

Detection of Anti-ENA and anti-dsDNA Antibodies Using Line Immunoassay in Systemic Autoimmune Diseases / 대한진단검사의학회지

BACKGROUND: Detection of antibodies to extractable nuclear antigens (ENAs) and dsDNA is needed for the diagnosis of and predicting prognosis in systemic autoimmune diseases. Recently introduced line immunoassay (LIA) has the advantage of detecting several autoantibodies simultaneously, and we evaluated its usefulness in the diagnosis of autoimmune diseases in comparison with enzyme-linked immunosorbent assay (ELISA). METHODS: Samples were collected from 437 patients referred by rheumatologists. FANA (fluorescent antinuclear antibody) test and LIA for the detection of 13 different autoantibodies, including 6 ENAs and dsDNA were performed. LIA-positive samples for ENA or dsDNA antibodies were further tested with ELISA. Final diagnosis was made by rheumatologists according to the diagnostic criteria. Agreement of results between LIA and ELISA was analyzed in 53 selected patients with systemic autoimmune diseases. RESULTS: The LIA detected antibodies to ENA and dsDNA in 118 and 22 patients, respectively, and ELISA detected 70.3% (83/118) and 45.5% (10/22) of LIA positive samples. Especially, 60.2% (71/118) of patients with positive ENA antibody on LIA was diagnosed as systemic autoimmune diseases. Patients having strong FANA titer and homogenous/speckled pattern showed higher prevalence of autoantibodies, but a small proportion of FANA negative patients also showed positive reactivity (LIA 10.8%, ELISA 5.2%). LIA showed a good agreement with ELISA for the anti-ENA antibodies (> or =80%), and a lower agreement for the anti-dsDNA antibody (67.9%). CONCLUSIONS: LIA detecting several autoantibodies simultaneously might replace ELISA for anti-ENA antibodies, but not for anti-dsDNA antibodies. When LIA is performed considering clinical manifestations and FANA, it could contribute to the diagnosis of systemic autoimmune disease.

Estudo do efeito de distintos períodos de fixação em formalina e métodos de recuperação antigênica na técnica de imuno-histoquímica / Study of the effect of different fixation times in formalin and methods of antigen retrieval in immunohistochemistry

Avaliou-se a influência do tempo de fixação em formalina neutra tamponada a 10 por cento e dos diferentes métodos de resgate de sítio antigênico induzido pelo calor [heat induced epitope retrieval (HIER)] para a imunoexpressão dos anticorpos anti-antígeno nuclear de proliferação celular (PCNA) e anti-AE1AE3 (citoqueratinas), empregados por apresentarem imunocoloração nuclear e citoplasmática ou submembranosa. Foram estudadas cinco tonsilas provenientes de amigdalectomias realizadas no Hospital São Paulo da Escola Paulista de Medicina da Universidade de São Paulo (UNIFESP/EPM), as quais foram seccionadas em 0,5cm² cada, fixadas em formalina, por períodos de tempo de seis, 12, 18, 24 e 48 horas e embebidas em parafina. Foram feitos cortes com 4mim em lâminas silanizadas. Para o estudo imuno-histoquímico utilizamos os anticorpos anti-PCNA e AE1AE3, empregando os três métodos de resgate de sítio antigênico: banho-maria, microondas e panela de pressão. A positividade na imunoexpressão do PCNA foi avaliada através da relação núcleos corados/total de núcleos x 100. A intensidade da coloração resultante foi avaliada através da utilização dos programas Corel Photo Paint 9 e UT Morph 2.0. Com relação à recuperação antigênica, concluímos que, para o anticorpo anti-PCNA no material fixado nos períodos de nosso estudo, os melhores resultados foram obtidos com o uso do microondas. O aumento do tempo de fixação interferiu na queda da imunopositividade do PCNA, em especial após o período de 24 horas. Em relação ao parâmetro intensidade de coloração para AE1AE3 nenhum dos métodos foi superior. A metodologia estudada para a análise semiquantitativa na intensidade da reação coincidiu com os resultados obtidos na avaliação criteriosa de cada uma das lâminas.