Name: performing and processing fna of anterior fat pad for amyloid
Uploaded: 2010-10-30T17:00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Agradecemos a la Sra. Bonnie Phetteplace, RN para la asistencia durante el vídeo de la demostración gráfica de procedimiento de la PAAF.

Introducción Amiloidosis sistémica es una enfermedad muy variable que hace referencia el depósito extracelular de proteínas diferentes que se conocen colectivamente como amiloide. La deposición de estas fibrillas de amiloide con la configuración plegada beta conduce a diferentes presentaciones clínicas en función de los órganos implicados. Las manifestaciones clínicas más relevantes de la amiloidosis sistémica se observan cuando hay participación de los órganos críticos como el corazón, el hígado y / o renal. Históricamente, estos órganos que se realizará una biopsia para demostrar la presencia de amiloide. Estos procedimientos moderadamente invasivos implicaba un riesgo importante, incluida la hemorragia. Aspiración de la almohadilla de grasa ha demostrado proporcionar un método confiable y no invasivo para la detección de amiloide en la amiloidosis sistémica 1. Este procedimiento es básicamente similar a la liposucción de la grasa subcutánea en la pared anterior del abdomen con anestesia local para recuperar el tejido fibroadiposo para la evaluación de los depósitos de amiloide en la escasa pequeñas paredes de los vasos sanguíneos 2. El método más frecuentemente utilizado para la identificación de amiloide en el tejido sigue siendo la característica de manzana verde que se ve cuando birrefringencia Congo secciones teñidas de rojo se visualizan con un microscopio de luz polarizada 3. Cuando la aspiración almohadilla de grasa se lleva a cabo, Congo manchas rojas se puede hacer en cualquiera de diapositivas directamente manchado o preparaciones de células bloque del tejido adiposo aspirado. Sin embargo, los pacientes en las primeras etapas de la amiloidosis tienen depósitos de amiloide escasa, lo que reduce enormemente la sensibilidad del Congo teñidas de rojo las secciones bloque de celdas 4,5. Evaluación ultraestructural de aspirados almohadilla de grasa por microscopía electrónica tiene una mejor reproducibilidad y la mejora de la sensibilidad 4. Por lo tanto, se recomienda a presentar todos los aspirados de la almohadilla de grasa, tanto para la preparación de un bloque de celdas y para la realización de microscopía electrónica 2. Aspiración de la almohadilla de grasa es un costo relativamente bajo y método no invasivo para la obtención de tejido para el diagnóstico de la amiloidosis sistémica. En este artículo se describe el procedimiento de aspiración de grasa almohadilla junto con detalles sobre el procesamiento de muestras para enviar muestras, tanto para la tinción con rojo y la evaluación ultraestructural por microscopía electrónica. En este vídeo, se demuestra este procedimiento reproducible y fácil de recuperar el material de diagnóstico óptimo. 1. Realización de la PAAF de la almohadilla de grasa anterior A. anestesia de la zona (Figura 1 y 2) <ol><li> Utilizando hisopos con alcohol o el agente de limpieza de la piel preferidos por una institución en particular, limpiar la piel en el área del cuadrante inferior del abdomen lateral a la línea media y por debajo del ombligo (Figura 1). </li><li> Marcar un área con forma de rombo de aproximadamente 2 x 2 pulgadas, como se muestra en la Figura 1, con un marcador. </li><li> Aspirar a unos 10 ml de lidocaína al 1% con una aguja 18G. Adjunte una aguja 25G de 1 ½ pulgadas de la jeringa. Eliminar el aire atrapado tocando la jeringa en posición vertical mientras se presiona el émbolo hasta que el líquido se dispensa y no hay burbujas de aire. </li><li> Anestesiar a lo largo de las fronteras de la zona ya marcada rombo, como se muestra en la Figura 2. Para empezar, la inserción de la aguja 25G debajo de la piel en el punto A y empuje con cuidado la aguja por vía subcutánea a punto X. Tire del émbolo de la jeringa para asegurarse de que no están dentro de un recipiente. A continuación, presione lentamente el émbolo para infiltrarse hasta 2,5 ml de lidocaína (alrededor de ¼ de la lidocaína en una jeringa de 10 ml) mientras se retira la aguja hasta el punto A, sin dejar la aguja sale de la piel en el punto A (figura 2a3). Con la aguja bajo la piel cambiar la dirección hacia el punto Y (figura 2a4) e introduzca la aguja por vía subcutánea a punto de Y (Figura 2b1). Al igual que antes, confirmar que la aguja no está en un recipiente y vierta aproximadamente 2,5 ml de lidocaína, mientras que poco a poco de retirar la aguja por el punto A. (Figura 2b3 través 2B4) Repita los pasos similares a partir del punto B y dispensación de lidocaína subcutánea de los puntos B y B a X a Y (Figura 2C1 través de 2d4). Evitar el sangrado en el aguijón A y B mediante la aplicación firme de la pieza de calibre estéril. </li><li> Ahora puede comprobar que la zona está anestesiada por tocar ligeramente la piel dentro del rombo anestesiados por la punta / rincón de la pieza de gasa de algodón, por comparación con la piel sin anestesia adyacentes. </li></ol> B. Realizar la aspiración almohadilla de grasa (Figura 3) (La aplicación de anestesia local antes de la ejecución de la aspiración de la almohadilla de grasa puede ser evitada, dependiendo de las preferencias regionales e individuales. Procedimiento correctamente realizado PAAF para la aspiración anterior almohadilla de grasa se puede completar en un pinchazo. Sin embargo, dependiendo del umbral de dolor de cada paciente , las maniobras de la aguja 18G de ida y vuelta en el tejido adiposo subcutáneo es relativamente distrescantar. El método descrito aquí anestesia para lograr el efecto en sólo dos pinchazos con una aguja 25G y evita el dolor con una mejor tolerancia al procedimiento.) <ol><li> Ensamble 18G 1 ½ pulgadas de aguja en una jeringa de 10 ml. Monte el conjunto de jeringa con aguja en las garras de la jeringa (&quot;agarre PAAF / pistola&quot;) para su correcta aplicación y liberación de vacío. </li><li> Inserte la punta de la aguja en la grasa subcutánea en el área romboidal limpia y anestesiados (Figura 3a). </li><li> Retraiga completamente el émbolo de la jeringa con la aguja que se inserta en el tejido subcutáneo para generar vacío (Figura 3b). </li><li> Mantener el vacío y la maniobra de la aguja hacia atrás y adelante en varias direcciones en la grasa subcutánea (Figura 3c a través de 3i). Cada golpe debe ser el mayor tiempo posible con la longitud de la aguja seleccionados sin dejar la aguja sale de la piel. Máximo de muestreo se logra cambiando de dirección con cada golpe (Figura 3i). Es importante mantener la dirección de la aguja tangencial a la serosa y en paralelo a la superficie de la piel para evitar la punción de la cavidad peritoneal. </li><li> El tejido fibroadiposo se acumula en la jeringa. Fragmentos de una vez suficiente tejido fibroadiposo (hasta 1 ml de muestra de fragmentos de sangre mezclada ricos) se recuperan, liberar el vacío y extraiga la aguja (Figura 3K). </li><li> Que el paciente o el asistente de aplicar una presión firme en el ámbito del procedimiento con una almohadilla de gasa para evitar la extravasación de sangre. </li></ol> 2. Procesamiento de muestras <ol><li> Lugar por lo menos 5-6 fragmentos de tejido fibroadiposo en solución de glutaraldehído para microscopía electrónica (fig. 4B). </li><li> Dependiendo del protocolo institucional, un frotis de algunos de los fragmentos de tejido fibroadiposo se puede preparar mediante la difusión de ellas entre dos diapositivas (Figura 4). </li><li> El material restante se deja coagular en la jeringa (puede tardar 5-7 minutos, dependiendo del tiempo de coagulación) (Figura 4 C). </li><li> Aspirar formalina al 10% en la jeringa para que el material del tejido fibroadiposo coagulada se desprendió de la pared de la jeringa y la libre flotación (Figura 4C2). Retire el émbolo de la jeringa (Figura 4C3) y transferir el tejido fibroadiposo coagulada en el recipiente de formol al 10% desde el extremo abierto de la jeringa frente al extremo de la boquilla (Figura 4C4). </li><li> Etiqueta de los envases debidamente y presentar el glutaraldehído para microscopía electrónica y el formol para H &amp; E y la sección de Congo mancha roja de evaluación en el marco de polarización microscopía. Si las manchas están preparados, pueden ser procesados ​​de acuerdo con el protocolo de cada laboratorio. </li></ol><img src="/files/ftp_upload/1747/1747fig1.jpg" alt="Figura 1" /> Figura 1. El área a ser anestesiado por PAAF de la almohadilla de grasa anterior. <img src="/files/ftp_upload/1747/1747fig2.jpg" alt="Figura 2" /> Figura 2. Anestesia de la zona. <img src="/files/ftp_upload/1747/1747fig3.jpg" alt="Figura 3" /> Figura 3. Realizar PAAF de la almohadilla de grasa anterior. <img src="/files/ftp_upload/1747/1747fig4.jpg" alt="Figura 4" /> Figura 4. Procesamiento de la almohadilla de grasa anterior aspirar a ser presentada al laboratorio para la detección de depósitos de amiloide. 3. Resultados representante <img src="/files/ftp_upload/1747/1747fig5.jpg" alt="Figura 5" /> Figura 5. Amiloides en la pared de los vasos sanguíneos pequeños en los fragmentos de tejido fibroadiposo. Tejido fibroadiposo fue procesado en formol, embebidos en parafina, se tiñeron con rojo congo, y examinadas por microscopía óptica de polarización. Amiloides en la pared de los vasos sanguíneos pequeños muestra birrefringencia verde manzana (flecha blanca). <img src="/files/ftp_upload/1747/1747fig6.jpg" alt="Figura 6" /> Figura 6. Los tejidos que carecen de la amiloidosis. La birrefringencia verde manzana está ausente en los tejidos de un paciente diferente sin amiloidosis. Birrefringencia azul (flecha blanca) de las fibras de colágeno, suelen estar presentes en casi todas las muestras. <img src="/files/ftp_upload/1747/1747fig7.jpg" alt="Figura 7" /> Figura 7. Fibrillas de amiloide en consonancia con la pared del vaso sanguíneo. Micrografía electrónica de recto, sin ramificaciones, esparcidas al azar 8-10 fibrillas nm de diámetro formado por amiloide en la pared del vaso sanguíneo.

<ol>
	<li>Westermark, P., Stenkvist, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+diagnosis+of+systemic+amyloidosis.">A new method for the diagnosis of systemic amyloidosis.</a> Arch Intern Med. 132, 522-523 (1973).</li><li>Devata, S., Hari, P., Markelova, N., Li, R., Shidham, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+AL+Amyloid+in+Abdominal+fat+pad+aspirates:+Is+Congo+red+sufficient?.">Detecting AL Amyloid in Abdominal fat pad aspirates: Is Congo red sufficient?.</a> Amyloid. , .</li><li>Merlini, G., Bellotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Amyloidosis.">Molecular Mechanisms of Amyloidosis.</a> New Engl J Med. 349 (6), 583-596 (2003).</li><li>Shidham, V. B., Kumar, N., Cihlar, K., Varsegi, G., Markelova, N., Li, R., Hari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+needle+aspiration+of+abdominal+fat+pad+for+diagnosis+of+early+amyloidosis:+How+can+the+clinical+role+of+the+test+be+improved?.">Fine needle aspiration of abdominal fat pad for diagnosis of early amyloidosis: How can the clinical role of the test be improved?.</a> Mod Pathol. 20, 1A-380A (2007).</li><li>Halloush, R., Lavrovskaya, E., Mody, D., Lager, D., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+typing+of+systemic+amyloidosis:+The+role+of+abdominal+fat+pad+fine+needle+aspiration.">Diagnosis and typing of systemic amyloidosis: The role of abdominal fat pad fine needle aspiration.</a> Cytojournal. 6, 24-24 (2010).</li><li>Hazenberg, I. I., Rijswijk, C. V. a. n., H, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Accuracy+of+Subcutaneous+Abdominal+Fat+Tissue+Aspiration+for+Detecting+Systemic+Amyloidosis+and+Its+Utility+in+Clinical+Practice.">Diagnostic Accuracy of Subcutaneous Abdominal Fat Tissue Aspiration for Detecting Systemic Amyloidosis and Its Utility in Clinical Practice.</a> Arthritis Rheum. , 54-546 (2006).</li><li>Guy, C. D., Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+fat+pad+aspiration+biopsy+for+tissue+confirmation+of+systemic+amyloidosis:+specificity,+positive+predictive+value,+and+diagnostic+pitfalls.">Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls.</a> Diagn Cytopathol. 24, 181-185 (2001).</li><li>Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+Amyloidosis.">Diagnosing Amyloidosis.</a> Scand J Rheumatol Suppl. 24, 327-329 (1995).</li><li>Linke, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Diagnosis+of+Amyloid+and+Various+Amyloid+Syndromes+Using+Congo+Red+Fluorescence.">Highly Sensitive Diagnosis of Amyloid and Various Amyloid Syndromes Using Congo Red Fluorescence.</a> Virchow Arch. 436, 439-448 (2000).</li><li>Giorgadze, T. A., Shiina, N., Baloch, Z. W., Tomaszewski, J. E., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+amyloid+in+fat+pad+aspiration:+an+evaluation+of+Congo+red+stain+by+fluorescent+microscopy.">Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.</a> Diagn Cytopathol. 31, 300-306 (2004).</li><li>DeMay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+Needle+Aspiration+Biopsy.">Fine Needle Aspiration Biopsy.</a> The Art &#38; Science of Cytopathology. , 465-46 (1996).</li><li>Arbustini, E., Vrga, L., Concardi, M., Pallandini, G., Obici, L., Merlini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+and+Immuno-electron+Microscopy+of+Abdominal+Fat+Identifies+and+Characterizes+Amyloid+Fibrils+in+Suspected+Cardiac+Amyloidosis.">Electron and Immuno-electron Microscopy of Abdominal Fat Identifies and Characterizes Amyloid Fibrils in Suspected Cardiac Amyloidosis.</a> Amyloid. 9, 108-114 (2002).</li></ol>

El diagnóstico de amiloidosis se logra generalmente con una biopsia de tejido de los órganos afectados, como el riñón, el hígado y / o del corazón. Este enfoque ha resultado del diagnóstico de alta, sin embargo, es invasivo y puede estar asociada con complicaciones como la hemorragia 2. Rectal, gingival y biopsias de médula ósea se prefiere para el diagnóstico como el enfoque relativamente menos invasivo en 1960 6,7,8. La aspiración de grasa abdominal se informó en el año 1973 como una caja fuerte, procedimiento mínimamente invasivo y sencillo, y menos costoso para el diagnóstico del tejido de la amiloidosis sistémica 1. Sin embargo, los detalles del procedimiento y el enfoque de procesamiento de la muestra recuperada no es coherente informó. Este vídeo y el artículo describe el procedimiento paso a paso. El enfoque para la detección de amiloide en la aspiración de la almohadilla de grasa, de manera similar, tampoco es bien estandarizados con unos pocos estudios comparar y evaluar los distintos enfoques 1,5,6,7,8. Evaluación de la anterior aspirados almohadilla de grasa con la tinción con rojo es menos sensible a la reproducibilidad interobservador baja, especialmente en los primeros casos de amiloidosis con depósitos de amiloide escasos 5. La inmunohistoquímica realizada en formol fija bloque de parafina de células secciones y Congo fluorescencia roja realizada en frotis citológico se han reportado para mejorar la sensibilidad diagnóstica de 9,10. La microscopía electrónica mejora la detección e identificación de las fibrillas de amiloide escaso en las pequeñas paredes de los vasos sanguíneos en el tejido fibroadiposo de la grasa aspira el parche 4, 5. Con base en esta información, este artículo se describe el procesamiento de la muestra para preparar los bloques de celdas (para la evaluación de rojo Congo secciones teñidas con bloque de celdas bajo el microscopio de polarización para el diagnóstico de manzana birrefringencia verde y también para la inmunohistoquímica, como se indica) y por microscopía electrónica. Sin embargo, dependiendo de la opción elegida para la evaluación de la muestra para el amiloide, que puede ser sometido a la preparación de muestra para citología, la preparación de bloques de células, y el procesamiento para microscopía electrónica. Algunos laboratorios realizan las pruebas en los frotis preparados a partir de los fragmentos de tejido fibroadiposo aspirado y se tiñen con rojo Congo para estudiar la fluorescencia 10. En general, en una amiloidosis tarde, el microscopio de luz con microscopía de rojo Congo polarización puede ser suficiente, sin embargo, en las primeras etapas de la enfermedad de las manchas de rojo Congo con microscopio de polarización en las secciones de bloque de celdas es menos sensible y fiable de la grasa aspirados pad 4,5,11 . Otras tinciones especiales como thioflavin T se puede utilizar con las mismas limitaciones. Otros enfoques para la detección de amiloide incluir la evaluación de los frotis de aspirado de amiloide con Congo microscopía de fluorescencia de color rojo polarizado y 10. En nuestra experiencia, este método es menos reproducible. Evaluación de los vasos sanguíneos en la almohadilla de grasa de amiloide mediante microscopía electrónica supera estas limitaciones 4,11. Además de la caracterización de amiloide ha sido reportado por inmunomicroscopía 12, sin embargo, estos métodos pueden no estar disponibles ampliamente en un centro de atención no terciaria. Las agujas utilizadas para la realización de procedimientos de aspiración se pueden clasificar según su tamaño calibre en finas (21-25G), intermedio (18-20G) y grandes (por ejemplo-14G) 11. Anterior aspiraciones almohadilla de grasa se ​​ha informado que se realiza mediante el uso de indicadores de variables que van desde la intermedia (18 a 20G) de multa (21 a 22G) 2,10. La mayoría de las lesiones tumorales son aspirados con agujas de calibre fino (por ejemplo, 25G) para obtener un buen frotis citológico, junto con pases adicionales con aguja de calibre amplio (por ejemplo, 18G) para recuperar muestras adicionales de los bloques de celdas. Durante la aspiración anterior almohadilla de grasa, el tejido fibroadiposo cohesionada no aspirar así con las agujas más finas. Preparación bloque de celdas y la microscopía electrónica son importantes para la evaluación de las pequeñas paredes de los vasos sanguíneos en los fragmentos de tejido fibroadiposo en el aspirado de grasa. Agujas de calibre amplio (por ejemplo, 18G) se recomiendan para obtener material diagnóstico adecuado 2,10. Como el procedimiento es comparable a la PAAF convencionales, la aspiración de la almohadilla de grasa que se conoce como la PAAF, aunque en general agujas de calibre puede ser utilizado para la realización de un 5.

A correction was made to <a href="http://www.jove.com/index/details.stp?id=1747">Performing and Processing FNA of Anterior Fat Pad for Amyloid</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Safwan S. Jaradeh
instead of:
Safwan S. Jardeh

Erratum: Performing and Processing FNA of Anterior Fat Pad for Amyloid

Torundas con alcohol, gasa, marcador, jeringas de 10 ml x 2, 1% de lidocaína (anestésico local, lidocaína Si se utiliza solo, se produce una sensación de ardor inicial después de la instilación. Esto se puede prevenir mediante el uso de una mezcla de 1:1 de 1% lidocaína y el 1% de sodio-bi-carbonato), calibre 18 (G) 1 ½ pulgadas de agujas x2 (para la realización de PAAF), 25G 1 ½ pulgadas de agujas (para inyectar un anestésico local), titular de la FNA jeringa (&quot;pistola&quot;), venda, vial de glutaraldehído (por microscopía electrónica), y el contenedor de la biopsia de formol al 10%.

performing and processing fna of anterior fat pad for amyloid

Históricamente, las biopsias de corazón, el hígado y el riñón se han realizado para demostrar los depósitos de amiloide en la amiloidosis. Desde la presentación clínica de esta enfermedad es muy variable y no específicos, los riesgos asociados de estas biopsias son demasiado grandes para el rendimiento diagnóstico. Otros sitios que tienen un menor riesgo de la biopsia, como la piel o gingival, son también relativamente invasivo y costoso. Además, estas biopsias no siempre pueden tener depósitos de amiloide suficiente para establecer un diagnóstico. Aspiración de la almohadilla de grasa ha demostrado una buena correlación clínica con un bajo costo y mínima morbilidad. Sin embargo, no existen protocolos estandarizados para la realización de este procedimiento o de procesamiento de la muestra de aspirado, lo que conduce a resultados variables y no reproducibles. La modalidad más utilizada para la detección de amiloide en los tejidos es una birrefringencia verde manzana en el Congo secciones teñidas de rojo con un microscopio de polarización. Esta técnica requiere una preparación de células bloque de material aspirado. Desafortunadamente, los pacientes que presentan en las primeras etapas de la amiloidosis tienen cantidades mínimas de amiloide, que reduce enormemente la sensibilidad del Congo teñidas de rojo las secciones bloque de celdas de aspirados almohadilla de grasa. Por lo tanto, la evaluación ultraestructural de aspirados almohadilla de grasa por microscopía electrónica debe ser utilizada, dada su mayor sensibilidad para la detección de amiloide. En este artículo se muestra un procedimiento simple y reproducible para la realización de la aspiración anterior almohadilla de grasa para la detección de amiloide, utilizando tanto la tinción con rojo de las secciones de bloque de celdas y la microscopia electrónica para la identificación ultraestructural.

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Performing and Processing FNA of Anterior Fat Pad for Amyloid

Aspiración de la almohadilla de grasa es un método preferido, el costo mínimamente invasiva, y baja en comparación con otros métodos para detectar amiloide para el diagnóstico de la amiloidosis sistémica. Este artículo vídeo muestra una descripción de los procedimientos para la realización de la aspiración almohadilla de grasa con el procesamiento adecuado de la muestra para el diagnóstico de los resultados óptimos.

Realización y procesamiento de la PAAF de almohadilla grasa anterior de amiloide

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

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Medicine

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

La purificación y la agregación de la proteína precursora amiloide del dominio intracelular

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Investigación

Medicina

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Procesamiento de mamoplastia de reducción y tejidos humanos Mastectomía para el cultivo celular

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Inyección ortotópico de células de cáncer de mama en el Mamaria Fat Pad de ratones para estudiar el crecimiento del tumor.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Evaluación de la viabilidad de la inyección de grasa humana en ratones desnudos con micro-tomografía computarizada

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Trasplante en la almohadilla del ratón de ovario grasa

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

La confirmación de la isquemia miocárdica y reperfusión en ratones utilizando electrocardiografía de superficie del cojín

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

Estroma Vascular fracción enriquecida Lipo para el tratamiento de neuromas de extremo sintomáticos

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

Metodología para la inducción de esputo y el procesamiento de laboratorio

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

Estrategias de muestreo y procesamiento de las muestras de tejido de Biobanco de porcinos modelos biomédicos

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Un sistema de entrenamiento y pruebas para la realización de reconstrucción vascular en Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...