**Accurate and High Sensitivity Identification of PNH Clones by Flow Cytometry Clones by Flow Cytometry**

**Accurate and High Sensitivity Identification of PNH** 

DOI: 10.5772/intechopen.71286

Iuri Marinov, Andrea Illingworth and D. Robert Sutherland D. Robert Sutherland Additional information is available at the end of the chapter

Iuri Marinov, Andrea Illingworth and

Additional information is available at the end of the chapter

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

#### **Abstract**

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

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,

**Dr. Marica Gemei**

Chelonia SA, Switzerland

leaving me free to explore all the potential and to try something new every day.

support flow cytometry development and its widespread use.

VIII Preface

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 laboratories with little prior experience performing PNH testing.

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