Ringraziamo Jessica Jones, Erica Clark, Costanza Ducar, Donna Smith, Roy Lewis, Lily Apedaile, Joanne Wiesner, Devin Adams, Corey McBain e Stephen Voght per la loro assistenza nello sviluppo di questo manoscritto metodo, e video. Questo lavoro è stato sostenuto dalla Fondazione Bill e Melinda Gates Foundation CAVD concessione 38645 (MJM) e National Institutes of concede Salute UM1 AI068618 e U01 AI069481 (MJM). EA-N. è sostenuto da NIH Grant T32 AI007140. Ringraziamo il James B. Charitable Trust Pendleton per la loro donazione generoso equipaggiamento.

Nota: per proteggere i fluoroforo anticorpi coniugati dalla luce, eseguire tutte le fasi di un bio-Armadio di sicurezza con la luce spenta. 1. Antibody colorazione Preparazione del pannello <ol><li> Il pannello colorazione anticorpo può essere trovato in Tabella 1. Concentrazione di anticorpi dovrebbe essere definita mediante titolazione con sangue intero e utilizzando la stessa attrezzatura citometria di flusso e le procedure che saranno utilizzati per acquisire i campioni fenotipizzazione macchiati. </li><li> Una volta che i titoli di colorazione adeguati sono determinati, unire tutti gli anticorpi in una miscela unica in un lock-tappo del tubo. Aggiungere tampone di lavaggio flusso (Dulbecco PBS con 2% di siero fetale bovino inattivato) per portare il volume totale di 100 microlitri. Scala la miscela per il numero di campioni che colorati. Questa miscela può essere conservato a 4 ° C per un massimo di otto settimane. </li></ol> 2. Colorazione <ol><li> Se il sangue raccolto deve essere utilizzato per altri scopi in annunciodizione di questo saggio, impostare una aliquota parte mentre più sensibili al tempo procedure sono eseguite il sangue residuo. L&#39;aliquota può essere conservato a temperatura ambiente per 4 ore dopo l&#39;iniezione in vena senza significativa perdita di cellule. </li><li> Verificare che non vi è un intatto tallone pellet sul fondo del tubo Trucount ed etichetta il tubo per identificare il campione viene macchiato. Negli studi clinici HVTN, il Laboratorio di Data Management System (Frontier Science and Technology Research Foundation, Amherst, NY) è usato per etichettare e tenere traccia dei campioni colorati. </li><li> Registrare i numeri di lotto e le date di scadenza di tutti i reagenti. Registrare il numero di conteggio Trucount tubo cordone fornito dal produttore sulla borsa di tubi, assicurarsi che il numero di lotto sulla borsa corrisponde al numero di lotto sul tubo. </li><li> Utilizzare pipettaggio inverso per pipettare esattamente 100 pl di sangue intero nella provetta Trucount, appena sopra il fermo metallico. Evitare sbavature di sangue lungo il lato del tubo. </li><li&gt; Uso regolare (in avanti) tecnica di pipettaggio, pipettare 100 pl del pannello misto colorazione anticorpale (Vedi Tabella 1) nel tubo Trucount. Tappare la provetta e vortex a bassa velocità per circa 15 secondi per mescolare. Ispezionare visivamente il tubo per garantire che il pellet tallone è completamente sciolto. </li><li> Incubare la provetta Trucount per 15 minuti a temperatura ambiente (15-30 ° C) al buio. </li><li> Se necessario, diluire una aliquota di 10 × soluzione FACS Lisi a 1X con DIH 2 O. Aggiungere 900 microlitri 1 × soluzione FACS Lisi al tubo. </li><li> Tappare la provetta e agitare bene a bassa velocità per circa 15 secondi per mescolare. Spingere verso il basso il tappo saldamente in posizione di bloccaggio sul tubo e sigillare con la pellicola di laboratorio. </li><li> Conservare il tubo a -65 ° C a -95 ° C fino a quando il campione è pronto per la spedizione al laboratorio di analisi centrale o per analisi in casa. I campioni sono stabili in questa fase per almeno quattro settimane. Se la spedizione o il saggio immediatamentetamente, questo passaggio può essere omesso. </li></ol> 3. Spedizione Nota: Le istruzioni che seguono utilizzano un sistema di trasporto isolato da Saf-T-Pak, Inc. idoneo alla spedizione categoria B sostanze biologiche esenti secondo la International Air Transport Association e (IATA). Se l&#39;analisi dei campioni nella stessa posizione come la colorazione si è verificato, andare alla sezione 4. <ol><li> I campioni possono essere spediti immediatamente dopo colorazione o una volta che sono congelati a -65 ° C a -95 ° C. Avvolgere ogni tubo completamente in pellicola e mettere in casella campione macchiato. Posizionare la scatola macchiato campione all&#39;interno di una tenuta poli-borsa con materiale assorbente. </li><li> Posizionare la tenuta poly-bag e contenuto in un sacchetto di Tyvek e sigillare con meno aria possibile nel sacchetto. </li><li> Posizionare il pacchetto campione (campione all&#39;interno imballaggio secondario) all&#39;interno della scatola interna marrone. </li><li> Posizionare l&#39;interno marrone bbue all&#39;interno del torace polistirolo, fissandolo nella dentellatura per evitare che si spostino. </li><li> Riempire la cassa in polistirolo con ghiaccio secco (circa 8 kg) e posizionare il coperchio a tenuta sul petto. </li><li> Nastro saldamente il contenitore di spedizione e la nave come sostanza biologica, Categoria B (UN3373) con il ghiaccio proprio a secco (UN1845) le marcature; seguire IATA PI-650 istruzioni. </li><li> Al ricevimento, i campioni vengono conservati a -65 ° C a -95 ° C fino a che vengono analizzati. </li></ol> 4. Scongelamento e Citometria a Flusso analisi <ol><li> Togliere il campione macchiato dal freezer a scongelare a temperatura ambiente al buio prima di raccogliere il citofluorimetro. Se la raccolta di dati su più di un campione, standardizzare il processo per tutte le provette sfalsando scongelamento modo che i tubi non sono seduti a temperatura ambiente per più di 1 ora ciascuno. </li><li> I campioni devono essere acquisiti utilizzando un citofluorimetro quattro laser dotato di filtri adatti, come il BD LSRII. Utilizzare standard di calibrazione citometro e metodi di compensazione fluorescenza per i dati di raccolta 4. </li></ol> Nota: Non impostare soglie scatter scatter in avanti o di lato durante la raccolta 5. Perline Trucount può scendere al di sotto della soglia impostata più basso possibile per questi parametri che causano un sottoinsieme di perline di non essere contabilizzati durante l&#39;analisi. Se richiesto dallo strumento di fissare una soglia, impostare il più basso possibile Am Ciano soglia canale. Perché leucociti CD45 + colorati con il pannello sarà Am Ciano positivo, e perline Trucount anche fluorescenza nel Ciano Am canale, ciò dovrebbe consentire di tutti i dati pertinenti devono essere opportunamente raccolti. <ol start="3"><li> Agitare la provetta per 5 secondi prima di caricare sul citofluorimetro. Perline Trucount reagiscono molto in molti canali. Porta le perle al momento della raccolta, trovando la popolazione che è altamente doppio positivo per Cy5 PE e APC (i due colors che più facilmente distinguere le perle dalle celle può variare a seconda strumentazione). Selezionare la porta tallone come arresto del cancello, e registrare i dati fino a quando almeno 20.000 perle vengono acquisiti. </li><li> Analizzare i dati utilizzando un software appropriato, come FlowJo (Treestar; Ashland, OR). Figura 1 mostra lo schema di gating utilizzata per l&#39;analisi delle diverse popolazioni leucocitarie da un pareggio rappresentante sangue di controllo. </li></ol> 5. Calcoli Trucount <ol><li> Ogni tubo Trucount contiene un pellet liofilizzato di perline fluorescenti. Dopo l&#39;aggiunta di liquido al tubo e di vortex, le perle dovrebbe diventare equamente distribuiti in tutto il campione. Il numero di perle in pellet varia leggermente da lotto e si trova sulla custodia per i tubi. </li><li> Porta le perline Trucount e delle popolazioni di cellule, come mostrato in Figura 1 per determinare i conteggi di eventi per ogni popolazione. Confrontando il numero di eventi in il cancello tallone al numero totale di perline originariamente nel tubo consente di determinare il rapporto di campione prelevato, che può quindi essere utilizzato per determinare la concentrazione assoluta (cioè cellule / mL) per ogni popolazione. La seguente equazione può essere utilizzato per questo scopo: La concentrazione cellulare (# cellule / pl sangue intero) = [# di popolazione (eventi / eventi / # # perlina perline totali in pellet)] / 100 ul. </li></ol> 6. Risultati rappresentativi <img src="/files/ftp_upload/4302/4302fig1.jpg" alt="Figura 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4302/4302fig1highres.jpg" /> Figura 1. Regime Soppressione utilizzato per l&#39;analisi delle principali popolazioni leucocitarie che mostrano i dati rappresentativi di un volontario sano. A) perline Trucount (i) sono gated ed esclusi dalle cellule. Granulociti (ii) sono delineati e lympohcytes e monociti sono divisi in 3 popluations: CD14linfociti negativi (iii), tutte le cellule CD14 negative (iv), e non-linfociti (v). B) CD14 linfociti negativi sono gated per distinguere cellule T CD4 + (vi), cellule T CD8 + (vii), cellule B (viii), CD56 cellule luminose NK (ix), CD56 cellule dim NK (x), e CD56 cellule negative NK (xi). C) Tutte le cellule CD14 negative sono gated distinguere mieloide (xii) e plasmacitoidi (xiii) cellule dendritiche. D) non-lympocytes sono gated di distinguere non classici (xiv), intermedi (xv), e classica (xvi) monociti. <a href="http://www.jove.com/files/ftp_upload/4302/4302fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . Sottopopolazioni cellulari più specifiche che non sono mostrati nella Figura 1 (ad esempio, cellule NKT o neutrofili) può anche essere distinti utilizzando il pannello presentiamo, e lo schema di gating può essere esteso o modificato per soddisfare esigenze specifiche di studio. Alcuni passi gating indicati sono unici a questo metodo. Di particolare nota, un cancello inclusione ecancello esclusione rimangono attorno alle perline Trucount e posti uno sopra l&#39;altro, uno per cancello per contare le perline, e uno di escludere le perline dall&#39;analisi cellulare (Figura 1A). Inoltre, poiché i linfociti, monociti e granulociti non sono così facilmente distinguibili nel sangue intero in quanto sono nelle cellule mononucleari del sangue periferico da scatter in avanti e laterale scatter, gating queste cellule con CD45 espressione e side scatter è spesso necessario (Figura 1A). Granulociti contaminanti (cerchio) che non potevano essere separati da linfociti e monociti con CD45 e side scatter si distinguono in alcuni appezzamenti per la loro alta espressione CD16 (Figura 1C e 1D Figura). Il numero di granulociti contaminanti è generalmente piccolo, e non interferiscono con gating monociti e cellule NK.

<ol>
	<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. Immunol. 143, 329-337 (2006).</li><li>Hosmalin, A., Lichtner, M., Louis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+analysis+of+dendritic+cell+subsets:+the+dendritogram.">Clinical analysis of dendritic cell subsets: the dendritogram.</a> Methods Mol. Biol. 415, 273-290 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

In questo rapporto, presentiamo una perlina metodo basato per l&#39;enumerazione di popolazioni leucocitarie in sangue fresco intero mediante citometria di flusso e coprire i parametri necessari per il suo utilizzo in uno studio clinico multicentrico, con l&#39;analisi del campione centralizzata. Questo metodo si basa su e ottimizza il protocollo BD Trucount e consente il suo utilizzo affidabile in un ambiente clinico multicentrico. Il saggio colorazione è semplice e richiede circa 45 minuti per eseguire, rendendo cos?...

<table border="1"><tbody><tr><td> Reagente Nome </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Trucount assoluti Tubi di conteggio </td><td> BD Biosciences </td><td> 340334 </td></tr><tr><td> FACS 10X Soluzione di Lisi </td><td> BD Biosciences </td><td> 349202 </td></tr><tr><td> Categoria B &amp; Sistema spedizione Esente, isolato </td><td> Saf-T-Pak </td><td> STP-320 </td></tr><tr><td> AmCyan CD45 anticorpo monoclonale </td><td> BD Biosciences </td><td> 339192 </td></tr><tr><td> CD3 FITC anticorpo monoclonale </td><td> BD Biosciences </td><td> 349201 </td></tr><tr><td> CD8 PerCP-Cy 5,5 anticorpo monoclonale </td><td> BD Biosciences </td><td> 341051 </td></tr><tr><td> CD4 Alexa Fluor 700 anticorpi monoclonalidy </td><td> BD Biosciences </td><td> 557922 </td></tr><tr><td> HLA-DR ECD anticorpo monoclonale </td><td> Beckman Coulter </td><td> IM3636 </td></tr><tr><td> V450 CD14 anticorpo monoclonale </td><td> BD Biosciences </td><td> 560349 </td></tr><tr><td> CD19 PE anticorpo monoclonale </td><td> BD Biosciences </td><td> 555413 </td></tr><tr><td> CD16 APC-H7 anticorpo monoclonale </td><td> BD Biosciences </td><td> 560195 </td></tr><tr><td> CD56-PE Cy7 anticorpo monoclonale </td><td> BD Biosciences </td><td> 335791 </td></tr><tr><td> CD11c APC anticorpo monoclonale </td><td> BD Biosciences </td><td> 559877 </td></tr><tr><td> CD123 PE-Cy5 anticorpo monoclonale </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

Crioconservazione dei leucociti del sangue periferico è ampiamente usato per conservare le cellule per la valutazione della risposta immunitaria negli studi clinici e offre molti vantaggi per la facilità e la standardizzazione delle valutazioni immunologiche, ma gli effetti negativi di questo processo sono stati osservati in alcuni sottogruppi di cellule, come i granulociti, cellule B , e cellule dendritiche 1-3. Saggiare leucociti freschi fornisce un quadro più preciso della stato in vivo delle cellule, ma è spesso difficile da eseguire nel contesto di ampi studi clinici. Saggi su cellule fresche sono dipendenti da impegni di volontariato e tempi e, se in termini di tempo, la loro applicazione può essere poco pratico a causa delle ore di lavoro richieste del personale di laboratorio. Inoltre, quando le sperimentazioni sono condotte a molteplici centri, laboratori con le risorse e la formazione necessarie per eseguire i saggi non può essere situato in prossimità sufficiente a siti clinici. Per risolvere questi problemi, abbiamo sviluppatopato un 11-colore del pannello colorazione anticorpo che può essere utilizzato con tubi Trucount (Becton Dickinson, San Jose, CA) e al fenotipo enumerare le popolazioni principali leucociti nel sangue periferico, producendo più robusto tipo cellulare informazioni specifiche di saggi come un emocromocitometrico completo (CBC), o con saggi disponibili in commercio pannelli progettati per tubi Trucount che macchiano solo per alcuni tipi di cellule. La procedura di colorazione è semplice, richiede solo 100 ml di sangue intero fresco, e dura circa 45 minuti, rendendo così possibile la standard di sangue laboratori di trasformazione da eseguire. Si è adattato dal tubo BD scheda tecnica Trucount ( <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">versione 8/2010</a> ). Il cocktail di anticorpi colorazione può essere preparata in anticipo in massa a un laboratorio saggio centrale e spediti a laboratori di lavorazione del sito. Tubi colorati possono essere fissatie congelati per la spedizione al laboratorio analisi centrale per l&#39;analisi di citometria a flusso multicolore. I dati generati da questo pannello colorazione può essere utilizzato per monitorare variazioni di concentrazione dei leucociti nel tempo in relazione all&#39;intervento e potrebbe facilmente essere ulteriormente sviluppato per valutare gli stati di attivazione di specifici tipi cellulari di interesse. In questo rapporto, dimostriamo la procedura utilizzata dal sangue di elaborazione tecnici di laboratorio per eseguire la colorazione di sangue intero fresco e la procedura per analizzare questi campioni macchiati in un laboratorio analisi centrale di supporto di uno studio clinico multicentrico. I dettagli del video la procedura in quanto viene eseguito nel contesto di un progetto di sperimentazione clinica del sangue nel HIV Vaccine Trials 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

In questo rapporto, dimostriamo la procedura di colorazione e l&#39;analisi di un test di fenotipizzazione effettuato su sangue intero fresco per enumerare le principali popolazioni innata e adattiva dei leucociti. Sottolineiamo considerazioni per l&#39;esecuzione di queste procedure nel contesto di uno studio clinico multicentrico.

Enumerazione delle popolazioni dei leucociti del sangue periferico per le principali studi clinici multicentrici utilizzando un intero saggio Fenotipizzazione Sangue

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

enumeration-major-peripheral-blood-leukocyte-populations-for

Research

JoVE Journal

Immunology and Infection

14.9K Views.  Fred Hutchinson Cancer Research Center. In questo rapporto, dimostriamo la procedura di colorazione e l'analisi di un test di fenotipizzazione effettuato su sangue intero fresco per enumerare le principali popolazioni innata e adattiva dei leucociti. Sottolineiamo considerazioni per l'esecuzione di queste procedure nel contesto di uno studio clinico multicentrico.

Video: 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.

L&#39;esaurimento delle popolazioni di cellule specifiche da deplezione del complemento

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

Ricerca

Immunologia e infezioni

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.

Purificazione della popolazione di cellule specifiche per fluorescenza separazione delle cellule attivate (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.

Espansione, di purificazione, e la valutazione funzionale delle cellule del sangue umano periferico NK

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.

Espansione del sangue periferico umano γδ cellule T con Zoledronato

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.

L&#39;introduzione di shear stress nello studio di adesione batterica

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 test funzionale di sangue intero per misurare la vitalità dei micobatteri, utilizzando Reporter-Gene Tagged BCG o 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.

La valutazione rapida e specifica del alogenazione perossidasi attività in campioni di sangue leucociti arricchita

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.

L&#39;uso di un monociti monostrato test per valutare fagocitosi Fcy recettore-mediata

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.

Destrano migliora l'efficienza di trasduzione lentivirale di cellule NK primarie Murine ed umane

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.

Cella singola analisi di Immunophenotype e produzione di citochine nel sangue periferico intero tramite citometria a massa

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