Nous tenons à remercier Jessica Jones, Erica Clark, Constance Ducar, Donna Smith, Roy Lewis, Lily Apedaile, Joanne Wiesner, Devin Adams, Corey McBain et Stephen Voght pour leur aide dans l&#39;élaboration de cette méthode, manuscrits et vidéo. Ce travail a été soutenu par la Fondation Bill et Melinda Gates CAVD subvention 38 645 (MJM) et les Instituts nationaux de la santé de subventions UM1 AI068618 et AI069481 U01 (MJM). EA-N. est soutenu par des subventions du NIH T32 AI007140. Nous remercions le James B. Pendleton Charitable Trust pour leur don généreux équipement.

Remarque: Pour protéger les anticorps conjugués fluorophore de la lumière, effectuer toutes les étapes d&#39;une armoire de biosécurité avec la lumière éteinte. 1. Préparation du panneau anticorps présentent une coloration <ol><li> Le panneau de coloration des anticorps peuvent être trouvés dans le tableau 1. Concentration d&#39;anticorps doivent être définis par titrage avec du sang total et en utilisant le matériel de cytométrie en flux même et procédures qui seront utilisées pour acquérir des échantillons de phénotypage tachés. </li><li> Une fois les titres de coloration appropriées sont déterminées, mélanger tous les anticorps dans un seul mélange dans un tube lock-bouchon. Ajouter tampon de lavage débit (PBS de Dulbecco avec inactivé à la chaleur 2% de sérum de veau foetal) pour porter le volume total à 100 ml. L&#39;échelle du mélange pour le nombre d&#39;échantillons colorés. Ce mélange peut être conservé à 4 ° C pendant un maximum de huit semaines. </li></ol> 2. Tachant <ol><li> Si le sang prélevé doit être utilisé à d&#39;autres fins en annoncecondition à cet essai, mis de côté pendant une aliquote plus sensibles au temps procédures sont effectuées sur le sang restant. L&#39;aliquote peut être conservé à température ambiante pendant 4 heures après la ponction veineuse sans perte significative de cellules. </li><li> Vérifier l&#39;existence d&#39;un bourrelet intact culot au fond du tube et Trucount étiquette du tube pour identifier l&#39;échantillon est coloré. Dans les essais cliniques HVTN, le Laboratoire de gestion des données du système (Frontier Science et de la Technologie Research Foundation, Amherst, New York) est utilisé pour marquer et suivre les échantillons colorés. </li><li> Notez les numéros de lot et les dates d&#39;expiration de tous les réactifs. Notez le numéro de tube Trucount nombre bourrelet prévu par le fabricant sur le sac de tubes, assurez-vous que le numéro de lot sur le sac correspond au numéro de lot sur le tube. </li><li> Utiliser reverse pipetage avec précision pipette 100 ul de sang total dans le tube Trucount, juste au-dessus de la retenue en métal. Éviter sang bavures sur le côté du tube. </li><li&gt; Utilisation régulière (marche avant) technique de pipetage, pipette 100 ul du panneau de coloration des anticorps mixte (voir tableau 1) dans le tube Trucount. Boucher le tube et agiter au vortex à basse vitesse pendant environ 15 secondes pour mélanger. Inspecter visuellement le tube de sorte que le culot de billes est entièrement dissous. </li><li> Incuber le tube Trucount pendant 15 minutes à température ambiante (15-30 ° C) dans l&#39;obscurité. </li><li> Si nécessaire, diluer une partie aliquote de 10 x solution de lyse FACS à l&#39;aide 1X diH 2 O. Ajouter 900 pl 1 x solution de lyse FACS sur le tube. </li><li> Boucher le tube et vortexer soigneusement à faible vitesse pendant environ 15 secondes pour mélanger. Appuyer sur le bouchon fermement en position de verrouillage sur le tube et le joint avec le film de laboratoire. </li><li> Stocker le tube à -65 ° C à -95 ° C jusqu&#39;à ce que l&#39;échantillon est prêt pour l&#39;expédition au laboratoire central d&#39;analyse pour l&#39;analyse ou dans la maison. Les échantillons sont stables à ce stade pour au moins quatre semaines. En cas d&#39;envoi ou de dosage immédiatediatement, cette étape peut être omise. </li></ol> 3. Transport maritime Remarque: Les instructions suivantes utilisent un système d&#39;expédition isolé de Saf-T-Pak, Inc spécialement conçu pour expédition de catégorie B exemptés des substances biologiques selon l&#39;International Air Transport Association et (IATA). Si l&#39;analyse des échantillons dans le même emplacement que la coloration produite, passez à la section 4. <ol><li> Les échantillons peuvent être expédiés immédiatement après coloration ou une fois qu&#39;ils sont congelés à -65 ° C à -95 ° C. Envelopper chaque tube complètement en aluminium et les placer dans la boîte d&#39;échantillons colorés. Placez la boîte dans un échantillon coloré étanche poly-sac avec un matériau absorbant. </li><li> Placez le poly-sac étanche et contenu dans un sac de Tyvek et le joint avec le moins d&#39;air possible dans le sac. </li><li> Placez le paquet d&#39;échantillon (échantillon à l&#39;intérieur de l&#39;emballage secondaire) à l&#39;intérieur de la boîte intérieure brun. </li><li> Placez le brun intérieure bbœuf à l&#39;intérieur de la poitrine en styromousse, en le fixant dans le renfoncement pour éviter tout déplacement. </li><li> Remplissez la poitrine polystyrène avec de la glace sèche (environ 8 kg) et placez le couvercle fermement sur la poitrine. </li><li> Fixez solidement le carton d&#39;expédition et navire comme substance biologique de catégorie B (3373) avec la glace sèche correcte (UN1845) les marquages; suivre l&#39;IATA PI-650 instructions. </li><li> Dès réception, les échantillons sont stockés à -65 ° C à -95 ° C jusqu&#39;à leur analyse. </li></ol> 4. Dégel et de l&#39;analyse de cytométrie en flux <ol><li> Retirer l&#39;échantillon coloré du congélateur pour décongeler à la température ambiante dans l&#39;obscurité avant de recueillir sur le cytomètre en flux. Si la collecte des données sur plus d&#39;un échantillon, de normaliser le processus pour tous les tubes en échelonnant la décongélation sorte que les tubes ne sont pas assis à la température ambiante pendant plus de 1 heure chacune. </li><li> Les échantillons doivent être acquises en utilisant un cytomètre de flux laser quatre équipés de filtres appropriés, tels que le LSR BDII. Utilisez l&#39;étalonnage cytomètre standard et les méthodes de compensation de fluorescence pour la collecte des données 4. </li></ol> Remarque: Ne réglez pas avant seuils de dispersion de dispersion ou de côté lors de la collecte 5. Perles Trucount peut tomber sous le seuil de réglage le plus bas possible pour ces paramètres causant un sous-ensemble de perles pour ne pas être pris en compte lors de l&#39;analyse. Si requis par l&#39;instrument de fixer un seuil, fixé le plus bas possible Am Cyan canal de seuil. Parce CD45 + leucocytes colorés avec le panneau sera Am Cyan positif, et des perles Trucount également une fluorescence dans le canal Am Cyan, ce qui devrait permettre à toutes les données pertinentes soient dûment collectées. <ol start="3"><li> Vortexer le tube échantillon pendant 5 secondes avant le chargement sur le cytomètre en flux. Perles Trucount fluorescence fortement dans de nombreux canaux. Porte des perles pendant la collecte en trouvant la population qui est très positif pour les deux PE Cy5 et l&#39;APC (la colo deuxrs que la plupart aisément distinguer les perles à partir des cellules peut varier en fonction de l&#39;instrumentation). Sélectionnez la porte de perles de votre porte d&#39;arrêt, et enregistrer des données jusqu&#39;à au moins 20.000 perles sont acquises. </li><li> Analyser les données en utilisant un logiciel approprié, comme FlowJo (Treestar, à Ashland, OR). Figure 1 montre le schéma de déclenchement utilisé pour l&#39;analyse des différentes populations leucocytaires d&#39;une prise de sang de contrôle représentant. </li></ol> 5. Calculs Trucount <ol><li> Chaque tube contient une Trucount pastille lyophilisée de billes fluorescentes. Après addition de liquide dans le tube et un vortex, les billes doivent être répartis également dans l&#39;échantillon. Le nombre de perles dans le culot varie légèrement selon le numéro de lot et peuvent être consultés sur le sac de rangement pour les tubes. </li><li> Porte des perles Trucount et des populations cellulaires comme le montre la figure 1 pour déterminer le nombre d&#39;événements pour chaque population. En comparant le nombre d&#39;événements in la porte du talon le nombre total de billes à l&#39;origine dans le tube vous permettra de déterminer le rapport de l&#39;échantillon collectés, qui peut ensuite être utilisée pour déterminer la concentration absolue (c.-à-cellules / ul) pour chaque population. L&#39;équation suivante peut être utilisée à cette fin: concentration cellulaire (cellules / ul # sang total) = [# / population événements (événements perles # / # perles totales en granulés)] / 100 pi. </li></ol> 6. Les résultats représentatifs <img src="/files/ftp_upload/4302/4302fig1.jpg" alt="Figure 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> Système d&#39;ouverture de porte Figure 1. Utilisés pour l&#39;analyse des principales populations de leucocytes contenant des données représentatives d&#39;un volontaire sain. A) Trucount perles (i) sont bloqués et exclues de cellules. Granulocytes (ii) sont délimitées et lympohcytes et les monocytes sont divisés en 3 popluations: CD14lymphocytes négatifs (iii), toutes les cellules CD14 négatives (iv), et non des lymphocytes (v). B) CD14 des lymphocytes négatifs sont déclenchés pour distinguer les cellules T CD4 (vi), lymphocytes T CD8 + (vii), les cellules B (viii), CD56 vives cellules NK (ix), CD56 dim cellules NK (x), et CD56 négatives des cellules NK (xi). C) Toutes les cellules CD14 négatives sont déclenchés à distinguer myéloïde (xii) et les cellules dendritiques plasmacytoïdes (xiii). D) Non-lympocytes sont déclenchés à distinguer non classiques (xiv), intermédiaires (xv), et classique (xvi) les monocytes. <a href="http://www.jove.com/files/ftp_upload/4302/4302fig1large.jpg" target="_blank">Cliquez ici pour agrandir la figure</a> . Sous-ensembles de cellules plus spécifiques qui ne sont pas représentés dans la figure 1 (par exemple, les cellules NKT ou neutrophiles) peuvent également être distingués en utilisant le panneau de nous présenter, et le système de déclenchement peuvent être élargis ou modifiés pour répondre aux besoins particuliers de l&#39;étude. Certaines étapes de déclenchement indiquées sont uniques à cette méthode. On notera en particulier, un portail d&#39;inclusion etgrille exclusion sont entraînés autour des talons Trucount et placé au-dessus de l&#39;autre, une grille à billes pour le comptage, et pour exclure une des perles de l&#39;analyse cellulaire (Figure 1A). Aussi, parce que les lymphocytes, les monocytes et les granulocytes ne sont pas aussi faciles à distinguer dans le sang entier car ils sont dans les cellules mononucléées du sang périphérique par diffusion vers l&#39;avant et le côté de dispersion, ouverture de porte à l&#39;aide de ces cellules CD45 expression et la diffusion latérale est souvent nécessaire (figure 1A). Granulocytes contaminants (encerclée) qui ne pouvaient pas être séparés de lymphocytes et les monocytes CD45 et utilisant la diffusion latérale se distinguent dans certaines parcelles de leur grande expression CD16 (figure 1C et 1D figure). Le nombre de granulocytes contaminants est généralement faible, et ils n&#39;interfèrent pas avec déclenchement cellulaire de monocytes et NK.

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	<li>Taylor, M. J., London, N. J., Thirdborough, S. M., Lake, S. P., James, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cryobiology+of+rat+and+human+dendritic+cells:+preservation+and+destruction+of+membrane+integrity+by+freezing.">The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing.</a> Cryobiology. 27, 269-278 (1990).</li><li>Reimann, K. A., Chernoff, M., Wilkening, C. L., Nickerson, C. E., Landay, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+lymphocyte+immunophenotype+and+proliferative+responses+in+cryopreserved+peripheral+blood+mononuclear+cells+from+human+immunodeficiency+virus+type+1-infected+donors:+implications+for+multicenter+clinical+trials.+The+ACTG+Immunology+Advanced+Technology+Laboratories.">Preservation of lymphocyte immunophenotype and proliferative responses in cryopreserved peripheral blood mononuclear cells from human immunodeficiency virus type 1-infected donors: implications for multicenter clinical trials. The ACTG Immunology Advanced Technology Laboratories.</a> Clin Diagn Lab Immunol. 7, 352-359 (2000).</li><li>Boonlayangoor, P., Telischi, M., Boonlayangoor, S., Sinclair, T. F., Millhouse, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+human+granulocytes:+study+of+granulocyte+function+and+ultrastructure.">Cryopreservation of human granulocytes: study of granulocyte function and ultrastructure.</a> Blood. 56, 237-245 (1980).</li><li>Perfetto, S. P., Ambrozak, D., Nguyen, R., Chattopadhyay, P., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+assurance+for+polychromatic+flow+cytometry.">Quality assurance for polychromatic flow cytometry.</a> Nat Protoc. 1, 1522-1530 (2006).</li><li>Brando, B., Barnett, D., Janossy, G., Mandy, F., Autran, B., Rothe, G., Scarpati, B., D'Avanzo, G., D'Hautcourt, J. L., Lenkei, R., Schmitz, G., Kunkl, A., Chianese, R., Papa, S., Gratama, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytofluorometric+methods+for+assessing+absolute+numbers+of+cell+subsets+in+blood.+European+Working+Group+on+Clinical+Cell+Analysis.">Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.</a> Cytometry. 42, 327-346 (2000).</li><li>Bull, M., Lee, D., Stucky, J., Chiu, Y. L., Rubin, A., Horton, H., McElrath, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+blood+processing+parameters+for+optimal+detection+of+cryopreserved+antigen-specific+responses+for+HIV+vaccine+trials.">Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials.</a> J. Immunol Methods. 322, 57-69 (2007).</li><li>Kantor, A. B., Roederer, M., Herzenberg, L., Blackwell, C., Weir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The handbook of Experimental Immunology. 43, 1-49 (1996).</li><li>Hulspas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+and+the+stability+of+phycoerythrin-tandem+dye+conjugates.">Flow cytometry and the stability of phycoerythrin-tandem dye conjugates.</a> Cytometry A. 75, 966-972 (2009).</li><li>Le Roy, C., Varin-Blank, N., Ajchenbaum-Cymbalista, F., Letestu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+APC-tandem+dyes+are+degraded+through+a+cell-dependent+mechanism.">Flow cytometry APC-tandem dyes are degraded through a cell-dependent mechanism.</a> Cytometry A. 75, 882-890 (2009).</li><li>Mandy, F., Brando, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+absolute+cell+counts+using+immunophenotypic+techniques.">Enumeration of absolute cell counts using immunophenotypic techniques.</a> Curr. Protoc. Cytom. Chapter 6, Unit 6 (2001).</li><li>Vuckovic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+dendritic+cells+in+clinical+practice+using+a+new+whole+blood+single-platform+TruCOUNT+assay.">Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay.</a> J. Immunol Methods. 284, 73-87 (2004).</li><li>Lichtner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+dendritic+cells+and+interferon-alpha+production+in+patients+with+tuberculosis:+correlation+with+clinical+outcome+and+treatment+response.">Circulating dendritic cells and interferon-alpha production in patients with tuberculosis: correlation with clinical outcome and treatment response.</a> Clin. Exp. 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Aucun conflit d&#39;intérêt déclaré.

Dans ce rapport, nous présentons une méthode basée sur cordon pour le dénombrement des populations de leucocytes dans le sang entier frais par cytométrie en flux et couvrir les paramètres nécessaires à son utilisation dans un essai clinique multicentrique avec analyse de l&#39;échantillon centralisée. Cette méthode s&#39;appuie sur le protocole et optimise BD Trucount et permet son utilisation fiable dans un cadre essai clinique multicentrique. L&#39;essai de coloration est simple et prend environ 45 minutes ...

<table border="1"><tbody><tr><td> Nom réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Trucount absolus Tubes de comptage </td><td> BD Biosciences </td><td> 340334 </td></tr><tr><td> FACS 10X solution de lyse </td><td> BD Biosciences </td><td> 349202 </td></tr><tr><td> Catégorie B &amp; système d&#39;expédition exonéré, isolé </td><td> Saf-T-Pak </td><td> STP-320 </td></tr><tr><td> CD45 anticorps monoclonal AmCyan </td><td> BD Biosciences </td><td> 339192 </td></tr><tr><td> CD3 FITC anticorps monoclonal </td><td> BD Biosciences </td><td> 349201 </td></tr><tr><td> CD8 PerCP-Cy 5,5 anticorps monoclonal </td><td> BD Biosciences </td><td> 341051 </td></tr><tr><td> CD4 Alexa Fluor 700 Antibo monoclonaldy </td><td> BD Biosciences </td><td> 557922 </td></tr><tr><td> HLA-DR DPE anticorps monoclonal </td><td> Beckman Coulter </td><td> IM3636 </td></tr><tr><td> CD14 anticorps monoclonal V450 </td><td> BD Biosciences </td><td> 560349 </td></tr><tr><td> CD19 PE anticorps monoclonal </td><td> BD Biosciences </td><td> 555413 </td></tr><tr><td> CD16 APC-H7 anticorps monoclonal </td><td> BD Biosciences </td><td> 560195 </td></tr><tr><td> CD56 PE-Cy7 anticorps monoclonal </td><td> BD Biosciences </td><td> 335791 </td></tr><tr><td> CD11c APC anticorps monoclonal </td><td> BD Biosciences </td><td> 559877 </td></tr><tr><td> CD123 PE-Cy5 anticorps monoclonal </td><td> BD Biosciences </td><td> 551065 </td></tr></tbody></table>

enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay

La cryoconservation des leucocytes du sang périphérique est largement utilisé pour conserver des cellules pour les évaluations de la réponse immunitaire dans les essais cliniques et offre de nombreux avantages pour la facilité et la standardisation des évaluations immunologiques, mais les effets néfastes de ce processus ont été observés sur certains sous-ensembles de cellules, telles que les granulocytes, les lymphocytes B , les cellules dendritiques et 1-3. Le dosage de leucocytes frais donne une image plus précise de l&#39;état dans vivo des cellules, mais il est souvent difficile à réaliser dans le cadre de vastes essais cliniques. Dosages de cellules fraîches dépendent des engagements volontaires et des délais, et si de temps, leur application peut être impossible en raison des heures de travail requis du personnel de laboratoire. En outre, lorsque les essais sont effectués dans plusieurs centres, les laboratoires avec les ressources et la formation nécessaires pour effectuer les dosages peuvent ne pas être situé à proximité suffisante de sites cliniques. Pour répondre à ces questions, nous avons développéloppé un panneau 11-couleur anticorps présentent une coloration qui peut être utilisé avec des tubes Trucount (Becton Dickinson, San Jose, CA) au phénotype et énumérer les principales populations de leucocytes dans le sang périphérique, ce qui donne plus robuste de type cellulaire renseignements précis que les tests comme un numération formule sanguine (CBC) ou des tests disponibles dans le commerce avec des panneaux conçus pour les tubes Trucount qui se colorent seulement pour quelques types cellulaires. La procédure de coloration est simple, ne nécessite que 100 ul de sang entier frais, et dure environ 45 minutes, ce qui permettrait aux standards du sang de traitement de laboratoires à effectuer. Il est adapté de la BD du tube Trucount fiche technique ( <a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">la version 8/2010</a> ). Le cocktail d&#39;anticorps coloration peut être préparé à l&#39;avance en vrac à un laboratoire d&#39;analyse central et expédiés aux laboratoires de traitement sur place. Stained tubes peuvent être fixéset congelé pour expédition au laboratoire d&#39;analyse central pour analyse par cytométrie en flux multicolore. Les données générées par ce panneau coloration peut être utilisé pour suivre l&#39;évolution des concentrations de leucocytes dans le temps par rapport à l&#39;intervention et pourrait facilement être développé afin d&#39;évaluer l&#39;état d&#39;activation des types de cellules spécifiques d&#39;intérêt. Dans ce rapport, nous démontrons la procédure utilisée par les techniciens de laboratoire de sang de traitement pour exécuter des taches sur du sang total frais et les mesures pour analyser ces échantillons colorés dans un laboratoire de test central supportant un essai clinique multicentrique. Les détails sur la vidéo de la procédure telle qu&#39;elle est pratiquée dans le cadre d&#39;un prélèvement sanguin essai clinique de vaccin contre le VIH pour les essais Network (HVTN).

Watch this Scientific Journal Video about Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay at JoVE.com

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Dans ce rapport, nous démontrons les étapes de coloration et de l&#39;analyse d&#39;un test de phénotypage réalisé sur sang total frais d&#39;énumérer les principaux innées et adaptatives des populations leucocytaires. Nous insistons sur des considérations d&#39;effectuer ces procédures dans le cadre d&#39;un essai clinique multicentrique.

Énumération des principales populations de leucocytes périphériques sanguins pour essais cliniques multicentriques en utilisant un test de sang total Phénotypage

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

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Immunology and Infection

14.9K vues.  Fred Hutchinson Cancer Research Center. Dans ce rapport, nous démontrons les étapes de coloration et de l'analyse d'un test de phénotypage réalisé sur sang total frais d'énumérer les principaux innées et adaptatives des populations leucocytaires. Nous insistons sur des considérations d'effectuer ces procédures dans le cadre d'un essai clinique multicentrique.

Vidéo: Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

Appauvrissement des populations de cellules spécifiques par l&#39;épuisement Complément

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

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Immunologie et infection

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Purification de population cellulaire spécifique par le tri cellulaire activé par fluorescence (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansion, purification et d&#39;évaluation fonctionnelle des cellules NK humaines périphérique Sang

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Expansion du sang périphérique humain cellules T γδ utilisant zolédronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

Présentation de contrainte de cisaillement dans l&#39;étude de l&#39;adhésion bactérienne

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Un dosage sanguin fonctionnelle entier pour mesurer la viabilité des mycobactéries, en utilisant Reporter-Gene Tagged BCG ou M. Tb (BCG Lux / M. Tb Lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

Évaluation rapide et spécifique de l&#39;halogénation peroxydase Activité dans les échantillons de sang enrichie en leucocytes

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Use of a Monocyte Monolayer Assay to Evaluate Fc&amp;#947; Receptor-mediated Phagocytosis

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Utilisation d&#39;un monocytes monocouche Assay pour évaluer phagocytose Fcy Receptor médiée

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextrane augmente l’efficacité de la Transduction des gènes des cellules NK primaires murines et humaines

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Analyse unicellulaires lymphoblastiques et Production de cytokines dans le sang périphérique par cytométrie de flux massique

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...