Name: enumeration of major peripheral blood leukocyte populations for multicenter clinical trials using a whole blood phenotyping assay
Uploaded: 2012-09-16T14:00:00
Description: 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

Vi takker Jessica Jones, Erica Clark, Constance Ducar, Donna Smith, Roy Lewis, Lily Apedaile, Joanne Wiesner, Devin Adams, Corey McBain og Stephen Voght for deres assistance i udviklingen af ​​denne metode, manuskript og video. Dette arbejde blev støttet af Bill og Melinda Gates Foundation CAVD tilskud 38.645 (MJM) og National Institutes of Health tilskud UM1 AI068618 og U01 AI069481 (MJM). EA-N. understøttes af NIH Grant T32 AI007140. Vi takker James B. Pendleton Charitable Trust for deres generøse udstyr donation.

Bemærk: For at beskytte fluorofor-konjugerede antistoffer mod lys, udføre alle trin i en bio-sikkerhed kabinet med lyset slukket. 1. Antistoffarvning Panel Preparation <ol><li> The antistoffarvning panel kan findes i tabel 1. Antistofkoncentration skal defineres ved titrering med fuldblod og anvendelse af samme flowcytometri udstyr og procedurer, der vil blive anvendt til at erhverve de farvede fænotypebestemmelse prøver. </li><li> Når passende farvning titer...

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I denne rapport præsenterer vi en perle-baseret metode til optælling leukocytpopulationer i frisk fuldblod ved flowcytometri og dække de parametre, der er nødvendige for dens anvendelse i et multicenter klinisk forsøg med centraliseret prøveanalyse. Denne metode bygger på og optimerer BD Trucount protokollen og sætter sin pålidelig brug i et multicenter klinisk forsøg indstilling. Farvningen assay er simpel og tager cirka 45 minutter at udføre, hvilket gør det muligt for blodbehandlingssystemer laboranter ti...

<table border="1"><tbody><tr><td> Reagens Name </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Trucount Absolute Counting Tubes </td><td> BD Biosciences </td><td> 340.334 </td></tr><tr><td> 10X FACS Lysing Solution </td><td> BD Biosciences </td><td> 349.202 </td></tr><tr><td> Kategori B &amp; Fritaget Shipping System, Isoleret </td><td> Saf-T-Pak </td><td> STP-320 </td></tr><tr><td> CD45 AmCyan monoklonalt antistof </td><td> BD Biosciences </td><td> 339.192 </td></tr><tr><td> CD3 FITC monoklonalt antistof </td><td> BD Biosciences </td><td> 349.201 </td></tr><tr><td> CD8 PerCP-Cy 5,5 monoklonalt antistof </td><td> BD Biosciences </td><td> 341.051 </td></tr><tr><td> CD4 Alexa Fluor 700 monoklonalt antibody </td><td> BD Biosciences </td><td> 557.922 </td></tr><tr><td> HLA-DR ECD monoklonalt antistof </td><td> Beckman Coulter </td><td> IM3636 </td></tr><tr><td> CD14 V450 monoklonalt antistof </td><td> BD Biosciences </td><td> 560.349 </td></tr><tr><td> CD19 PE monoklonalt antistof </td><td> BD Biosciences </td><td> 555.413 </td></tr><tr><td> CD16 APC-H7 monoklonalt antistof </td><td> BD Biosciences </td><td> 560.195 </td></tr><tr><td> CD56 PE-Cy7 monoklonalt antistof </td><td> BD Biosciences </td><td> 335.791 </td></tr><tr><td> CD11c APC monoklonalt antistof </td><td> BD Biosciences </td><td> 559.877 </td></tr><tr><td> CD123 PE-Cy5 monoklonalt antistof </td><td> BD Biosciences </td><td> 551.065 </td></tr></tbody></table>

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

Kryopræservering af perifere blodleukocytter er almindeligt anvendt til at bevare celler til immunreaktionsmodificerende i kliniske forsøg og giver mange fordele for nem og standardisering af immunologiske vurderinger, men skadelige virkninger af denne proces er blevet iagttaget nogle celledelmængder, såsom granulocytter, B-celler , og dendritiske celler 1-3. Analyse friske leukocytter giver et mere nøjagtigt billede af in vivo tilstanden af cellerne, men er ofte vanskeligt at udføre i forbindelse med store kliniske forsøg. Friske celleassays er afhængige frivillige forpligtelser og tidsrammer, og hvis tidskrævende, kan deres anvendelse være upraktisk på grund af de arbejdstimer, der kræves af laboratoriepersonale. Hertil kommer, at når der udføres forsøg på flere centre kan laboratorierne med de ressourcer og uddannelse er nødvendige for at udføre de analyser ikke være placeret i tilstrækkelig nærhed til kliniske steder. For at løse disse problemer, har vi udvikletviklet en 11-farve antistoffarvning panel, der kan anvendes med Trucount rør (Becton Dickinson, San Jose, CA) til fænotype og tælle de store leukocytpopulationer i perifert blod, hvilket giver mere robust celletypespecifik information end assays, såsom et komplet blodtælling (CBC) eller assays med kommercielt tilgængelige paneler designet til Trucount rør, der farves for kun et par celletyper. Farvningsproceduren er simpel, kræver kun 100 ul frisk fuldblod, og tager cirka 45 minutter, hvilket gør det muligt for almindelige blod-behandling labs at udføre. Det er tilpasset fra BD Trucount røret Technical Data Sheet ( <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">udgave 8/2010</a> ). Farvningen antistof cocktail kan fremstilles i forvejen i løs vægt på en central assay laboratorium og sendes til webstedet behandling labs. Farvede rør kan fastsættesog nedfrosset til forsendelse til den centrale assay laboratorium til flerfarvet flowcytometrianalyse. De data, der genereres fra denne farvning panel kan bruges til at spore ændringer i leukocyt koncentrationer over tid i forhold til intervention og let kunne videreudvikles til at vurdere aktivering tilstande af bestemte celletyper af interesse. I denne rapport, viser vi den procedure, der anvendes af blod-behandling laboratorieteknikere til at udføre farvning på frisk fuldblod og de skridt til at analysere disse farvede prøver på et centralt assay laboratorium støtter en multicenter klinisk forsøg. Den video detaljer proceduren, som den udføres i forbindelse med et klinisk forsøg blod uafgjort i 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

I denne rapport, viser vi de farvning og analyse trin i en fænotypebestemmelse assay udført på frisk fuldblod til at optælle de store medfødte og adaptive leukocytpopulationer. Vi lægger vægt på overvejelser for disse procedurer udføres i forbindelse med et multicenter klinisk forsøg.

Optælling af Major perifert blod leukocytpopulationer for multicenter kliniske undersøgelser med en Whole Blood fænotypebestemmelse Assay

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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