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

Мы благодарим Джессика Джонс, Эрик Кларк, Констанция Ducar, Донна Смит, Рой Льюис, Лили Apedaile, Джоан Визнер, Девин Адамс, Кори McBain и Стивен Voght за их помощь в развитии этого метода, рукописи и видео. Эта работа была поддержана Фондом Билла и Мелинды Гейтс грант CAVD 38645 (MJM) и Национального института здоровья гранты UM1 AI068618 и U01 AI069481 (MJM). EA-N. при поддержке гранта NIH T32 AI007140. Мы благодарим Джеймс Б. Пендлтон благотворительного фонда за их щедрые пожертвования оборудованием.

Примечание: Для защиты флуорофора-антителами от света, выполнить все этапы биологической безопасности шкаф с свет. 1. Антитела Окрашивание Подготовка панелей <ol><li> Панель антител окрашивания можно найти в таблице 1. Концентрации антител должно быть опреде...

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В данном отчете мы представляем шарик на основе метода для перечисления популяции лейкоцитов в свежей цельной крови методом проточной цитометрии и охватывают параметры, необходимые для его использования в многоцентровых клинических испытаний с централизованным анализом образцов. Э...

<table border="1"><tbody><tr><td> Реагент Имя </td><td> Компания </td><td> Номер в каталоге </td></tr><tr><td> Trucount Абсолютный подсчет труб </td><td> BD Biosciences </td><td> 340334 </td></tr><tr><td> 10X FACS Lysing Solution </td><td> BD Biosciences </td><td> 349202 </td></tr><tr><td> Категория B &amp; Освобожденный система доставки, изолированный </td><td> Saf-T-Pak </td><td> STP-320 </td></tr><tr><td> AmCyan CD45 моноклональных антител </td><td> BD Biosciences </td><td> 339192 </td></tr><tr><td> CD3 FITC моноклональных антител </td><td> BD Biosciences </td><td> 349201 </td></tr><tr><td> CD8 PerCP-Су 5,5 моноклональных антител </td><td> BD Biosciences </td><td> 341051 </td></tr><tr><td> CD4 Alexa Fluor 700 моноклональные Antiboду </td><td> BD Biosciences </td><td> 557922 </td></tr><tr><td> HLA-DR ECD моноклональных антител </td><td> Beckman Coulter </td><td> IM3636 </td></tr><tr><td> V450 CD14 моноклональных антител </td><td> BD Biosciences </td><td> 560349 </td></tr><tr><td> CD19 PE моноклональных антител </td><td> BD Biosciences </td><td> 555413 </td></tr><tr><td> CD16 APC-H7 моноклональных антител </td><td> BD Biosciences </td><td> 560195 </td></tr><tr><td> CD56 PE-Cy7 моноклональных антител </td><td> BD Biosciences </td><td> 335791 </td></tr><tr><td> CD11c APC моноклональных антител </td><td> BD Biosciences </td><td> 559877 </td></tr><tr><td> CD123 PE-Cy5 моноклональных антител </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

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube 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">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the 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

В этом докладе мы покажем, окрашивание и анализ шагов фенотипирование анализ выполняется на свежую цельную кровь, перечислить основные врожденной и адаптивной популяции лейкоцитов. Мы подчеркиваем соображения для выполнения этих процедур в рамках многоцентрового клинического исследования.

Перечень основных периферической крови популяции лейкоцитов для многоцентровых клинических испытаний с использованием полного анализа крови Фенотипирование

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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