Preface

In spite of flow cytometry being the gold standard in hematologic and oncohematologic re‐ search and diagnosis, its use in industrial drug discovery and research has been minimal for many years. Additionally, it has been used for simple and low content analysis, from cell cycle evaluation to transfection level control, often on homogeneous cell populations. This is because for many years it has not been a widespread technique in research laboratories and its use requires in-depth training and experience to gain reproducible and sound results. More recently, instruments have evolved that are now both stable and easy to use, and anal‐ ysis software has become user friendly and affordable, even for beginners. Industrial re‐ search has implemented flow cytometry in routinely used techniques because of its validation as a simple and cost-effective substitute for many assays, because of its radioli‐ gand bindings, and because of the strong automation achievable. Recently, various highthroughput flow cytometry screening applications have been reported in formats up to 1536. In addition to cell-based applications, there has also been an increase in performing highthroughput flow cytometry using bead-based immunoassays to screen for cytokines and other proteins in a multiplexed format.

Flow cytometry has been incorporated in research programs as a flexible and multipurpose technique to evaluate many biological responses and readouts with a single instrument. Flow cytometers allow high-speed analysis of particles (up to thousands of single cells per second) and measure multiparameters (up to 18) on single cells simultaneously. Because of its characteristics, flow cytometry can be used to perform high-throughput analysis but also high content screening. Complex tissues can be homogenized and treated as mixtures of cell lineages in a single run, while composing cell populations can be isolated later during the analysis allowing the visualization of an entire tissue and of the cell interactions and changes in different physiopathological conditions. Using cell barcoding techniques, differ‐ ent samples, treated in different ways or deriving from different tissues or experimental conditions, can be mixed and directly compared in a single run, further increasing the multi‐ parametric potential of flow cytometry.

Recently, the introduction of imaging flow cytometry has filled the gap between flow cy‐ tometry and imaging allowing the precise evaluation of visual parameters as a speckled dis‐ tribution of mutant proteins inside the cells, which is impossible to evaluate in conventional cytometry. Flow cytometry can be further exploited than it is today using the full potential of new instruments and new reagents and kits available for multiparameter and high con‐ tent analysis.

This book collects some examples of how different parameters can be combined and ana‐ lyzed to obtain a valuable analysis in different contexts. Some chapters describe applications in the diagnostic and prognostic field as common CD4 T-cell enumeration or more advanced analysis as PNH clone identification and cell-cell interaction analysis in the leukemia micro‐ environment. Other chapters treat the less widespread analysis of microalgae quality assess‐ ment, an emerging industrial need, or the use of flow cytometry in the testing of blood handling medical devices, thus showing that flow cytometry is not only applicable to re‐ search and clinical analyses. Finally, practical examples of how academically developed as‐ says can be optimized and standardized for industrial purposes have prompted all of us to support flow cytometry development and its widespread use.

The contributors to the book include flow cytometry users from both academia and industry with different needs and visions; the high diversity of flow cytometer applications is re‐ sumed by the presented diverse mix of expertise, a hallmark of the flow cytometry com‐ munity in which innovation is a driver. I wish to sincerely thank all the contributors for their expertise, personal vision and effort in the writing of this book. Thanks also to IntechOpen for approaching me about a project I really care about. Finally, thanks to Luigi Del Vecchio for introducing me to flow cytometry without the barrier of a formal education and training, leaving me free to explore all the potential and to try something new every day.

> **Dr. Marica Gemei** Chelonia SA, Switzerland

**Chapter 1**

**Provisional chapter**

**Accurate and High Sensitivity Identification of PNH**

Flow cytometry performs a key role in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Careful selection and validation of antibody conjugates have allowed the development of reagent cocktails suitable for the high sensitivity detection of PNH red blood cells (RBCs) and white blood cells (WBCs) in PNH and related diseases such as aplastic anemia (AA) and some subsets of myelodysplastic syndromes (MDS). A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at a limit of quantification (LOQ) of 0.01% or better. While separate 4-color Fluorescent Aerolysin (FLAER), CD24, CD15 and CD45-based neutrophil and FLAER, CD14, CD64 and CD45 based monocyte assays were developed to detect PNH WBC phenotypes, 5-, 6- and 7-color assays have subsequently been developed for more modern cytometers equipped with five or more fluorescence detectors. For instrumentation with five detectors, a single tube 5-color FLAER, CD157, CD15, CD64 and CD45-based assay to simultaneously detect PNH neutrophils and monocytes has been developed. For instruments with six or more detectors and multiple lasers, a variety of 5-, 6- and 7-color assays have been developed using combinations of FLAER, CD24, CD14 and CD157. All WBC assays have a limit of quantification (LOQ) of 0.1% or better. Using these standardized approaches, results have demonstrated good intra- and inter-laboratory performance characteristics even in

**Accurate and High Sensitivity Identification of PNH** 

DOI: 10.5772/intechopen.71286

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Paroxysmal nocturnal hemoglobinuria is a rare, life-threatening acquired hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidylinositol glycan

**Keywords:** PNH, flow cytometry, high sensitivity assay, validation, standardization

laboratories with little prior experience performing PNH testing.

**Clones by Flow Cytometry**

**Clones by Flow Cytometry**

Iuri Marinov, Andrea Illingworth and

Iuri Marinov, Andrea Illingworth and

http://dx.doi.org/10.5772/intechopen.71286

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

D. Robert Sutherland

**Abstract**

**1. Introduction**

D. Robert Sutherland

**Provisional chapter**
