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Clinical Immunology - Articles and news items
Flow Cytometry, Issue 2 2012 / 26 April 2012 / J. Paul Robinson, Purdue University Cytometry Laboratories & Weldon School of Biomedical Engineering, Purdue University; Bernd Bodenmiller, Group leader, Institute of Molecular Life Sciences, University of Zurich; Valery Patsekin and Bartek Rajwa, Purdue University Cytometry Laboratories, Purdue University; and V. J. Davisson, Medicinal Chemistry & Molecular Pharmacology, Purdue University
Flow cytometry is the technology that has the most impact on single-cell analysis. Over the past 40 years, it has arguably been the single most important research technique in the fields of basic and applied immunology. Flow cytometry excels in quantitative evaluation of receptor expression, separation of functionally defined cell populations and monitoring of cellular differentiation. For the clinical sciences, flow cytometry has been a key tool for diagnostics whereby aberrant populations are identified, classified and quantified, and in situations such as minimal residual disease, is capable of identifying rare cells indicative of dysplasia.
Despite the importance of its applications, flow cytometry is often seen as an aging technique without new exciting opportunities. The reality could not be further from the truth. The systems biology approach breathes new life into this unique technology. The new challenges of complex analysis of networks and pathways are a natural fit for modern multifactorial flow cytometry.
At the dawn of modern immunology, Len Herzenberg at Stanford University demonstrated the power of this high-tech tool in the early 1970s and it was soon the select tool for immunologists1. The advantage of flow cytometry was clear to most.
The process of building robust PCR/qPCR assays is a matter of perseverance and consistency. A few questions that should be answered prior to starting development will help make the process more efficient and effective:
Does the assay need to simply detect the presence of the target (qualitative), or must it assign a value to the detected target (quantitative)? The development process for a qualitative or quantitative assay, although similar in many respects, ultimately will take different paths
In what type of matrix will samples be? Matrix plays an important role in both development and validation of the assay. If the assay is needed for multiple matrices (whole blood, plasma, serum, differing tissue types, etc.), each matrix must be evaluated individually to determine its impact on assay performance
Will extraction be required, and by what method? 4) What throughput will be needed?
Answering these questions early in the process will help prevent ‘reworks’ later.
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