Summary

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

Published: September 16, 2012
doi:

Summary

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.

Abstract

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 μl 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 (version 8/2010). 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).

Protocol

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

Discussion

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…

Divulgations

The authors have nothing to disclose.

Acknowledgements

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.

Materials

Reagent Name Company Catalogue number
Trucount Absolute Counting Tubes BD Biosciences 340334
10X FACS Lysing Solution BD Biosciences 349202
Category B & Exempt Shipping System, Insulated Saf-T-Pak STP-320
CD45 AmCyan monoclonal antibody BD Biosciences 339192
CD3 FITC monoclonal antibody BD Biosciences 349201
CD8 PerCp-Cy 5.5 monoclonal antibody BD Biosciences 341051
CD4 Alexa Fluor 700 monoclonal antibody BD Biosciences 557922
HLA-DR ECD monoclonal antibody Beckman Coulter IM3636
CD14 v450 monoclonal antibody BD Biosciences 560349
CD19 PE monoclonal antibody BD Biosciences 555413
CD16 APC-H7 monoclonal antibody BD Biosciences 560195
CD56 PE-Cy7 monoclonal antibody BD Biosciences 335791
CD11c APC monoclonal antibody BD Biosciences 559877
CD123 PE-Cy5 monoclonal antibody BD Biosciences 551065

References

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Hensley, T. R., Easter, A. B., Gerdts, S. E., De Rosa, S. C., Heit, A., McElrath, M. J., Andersen-Nissen, E. Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay. J. Vis. Exp. (67), e4302, doi:10.3791/4302 (2012).

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