मेजर multicenter चिकित्सीय परीक्षण के लिए परिधीय रक्त ल्युकोसैट आबादी एक पूरे रक्त phenotyping परख का उपयोग कर की गणना
Summary
इस रिपोर्ट में, हम एक phenotyping परख प्रमुख जन्मजात और अनुकूली ल्युकोसैट आबादी की गणना के लिए नए सिरे से पूरे रक्त पर प्रदर्शन करने के धुंधला हो जाना और विश्लेषण कदम को प्रदर्शित करता है. हम एक multicenter चिकित्सीय परीक्षण के संदर्भ में इन प्रक्रियाओं के प्रदर्शन के लिए विचार पर जोर.
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
Discussion
इस रिपोर्ट में, हम प्रवाह cytometry द्वारा ताजा पूरे रक्त में ल्युकोसैट आबादी enumerating के लिए एक विधि मनका आधारित प्रस्तुत और मापदंडों केंद्रीकृत नमूना विश्लेषण के साथ अपने एक multicenter चिकित्सीय परीक्षण में उपयोग ?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
हम इस विधि पांडुलिपि, और वीडियो के विकास में उनकी सहायता के लिए जेसिका जोन्स, एरिका क्लार्क, Constance Ducar, डोना स्मिथ, रॉय लुईस, लिली Apedaile, Joanne Wiesner, Devin एडम्स, कोरी McBain और स्टीफन Voght धन्यवाद.
इस काम के बिल और मेलिंडा गेट्स फाउंडेशन CAVD अनुदान 38645 (MJM) और अनुदान स्वास्थ्य के राष्ट्रीय संस्थान AI068618 UM1 और AI069481 U01 (MJM) द्वारा समर्थित किया गया. EA-N. NIH अनुदान T32 AI007140 द्वारा समर्थित है. हम उनके उदार उपकरण दान के लिए जेम्स बी Pendleton चैरिटेबल ट्रस्ट धन्यवाद.
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 |
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